NMR device for detection of analytes

ABSTRACT

This invention relates generally to detection devices having one or more small wells each surrounded by, or in close proximity to, an NMR micro coil, each well containing a liquid sample with magnetic nanoparticles that self-assemble or disperse in the presence of a target analyte, thereby altering the measured NMR properties of the liquid sample. The device may be used, for example, as a portable unit for point of care diagnosis and/or field use, or the device may be implanted for continuous or intermittent monitoring of one or more biological species of interest in a patient.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/844,672, filed Jul. 27, 2010, which is a continuation of U.S. patentapplication Ser. No. 12/231,426, filed Sep. 2, 2008, which is acontinuation of U.S. patent application Ser. No. 11/513,503, filed Aug.31, 2006, now U.S. Pat. No. 7,564,245, which claims the benefit of U.S.Provisional Patent Application No. 60/713,176, filed Aug. 31, 2005, eachof which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to devices for the detection ofanalytes. More particularly, in certain embodiments, the inventionrelates to a detection device having one or more small wells eachsurrounded by, or in close proximity to, an NMR micro coil, each wellcontaining a liquid sample with magnetic nanoparticles thatself-assemble or disperse in the presence of a target analyte, therebyaltering the measured NMR properties of the liquid sample.

BACKGROUND OF THE INVENTION

Biocompatible magnetic nanosensors have been designed to detectmolecular interactions in biological media. Upon target binding, thesenanosensors cause changes in the spin-spin relaxation times ofneighboring water molecules (or any solvent molecule with freehydrogens) of a sample, which can be detected by classical magneticresonance (NMR/MRI) techniques. Thus, by using these nanosensors in aliquid sample, it is possible to detect the presence of an analyte atvery low concentration—for example, small molecules, specific DNA, RNA,proteins, carbohydrates, organisms, and pathogens (e.g. viruses)—withsensitivity in the low femtomole range (from about 0.5 to about 30fmol).

In general, magnetic nanosensors are superparamagnetic nanoparticlesthat bind or otherwise link to their intended molecular target to formclusters (aggregates) or nanoassemblies. It is thought that whensuperparamagnetic nanoparticles assemble into clusters and the effectivecross sectional area becomes larger, the nanoassembly becomes moreefficient at dephasing the spins of surrounding water (or other solvent)protons, leading to an enhancement of the measured relaxation rates(1/T2). Additionally, nanoassembly formation can be designed to bereversible (e.g., by temperature shift, chemical cleavage, pH shift,etc.) so that “forward” or “reverse” assays can be developed fordetection of specific analytes. Forward (clustering) and reverse(declustering) types of assays can be used to detect a wide variety ofbiologically relevant materials. Furthermore, the spin-latticerelaxation time (T1) is considered independent of nanoparticle assemblyformation and can be used to measure concentration in bothnano-assembled and dispersed states within the same solution.

Examples of magnetic nanosensors are described in Perez et al., “Use ofMagnetic Nanoparticles as Nanosensors to Probe for MolecularInteractions,” ChemBioChem, 2004, 5, 261-264, and in U.S. PatentApplication Publication No. US2003/0092029 (Josephson et at), the textsof which are incorporated by reference herein, in their entirety.Examples of magnetic nanosensors include monocrystalline iron oxidenanoparticles from about 3 to about 5 nm in diameter surrounded with adextran coating approximately 10 nm thick such that the averageresulting particle size is from about 25 to about 30 nm.

More stably coated and amino-functionalized nanosensors can be prepared,for example, by cross-linking the dextran coating of the metal oxideparticle core with epichlorohydrin, then treating with ammonia toprovide functional amino groups. Aminated cross-linked iron oxidenanoparticles (amino-CLIO) have been made with 40 amino groups perparticle, with an average particle size from about 40 to about 50 nm.These particles can withstand harsh treatment, such as incubation at120° C. for 30 minutes, without a change in size or loss of theirdextran coat. Amino groups in amino-CLIO can react byN-hydroxysuccinimide (NHS) based bifunctional cross-linking, allowingattachment of a range of sulfhydryl-bearing biomolecules. This givesrise to biomolecule-nanoparticle conjugates with unique biologicalproperties. In addition to their use as sensors, the resultantsuperparamagnetic nanoparticles are valuable for imaging specificmolecular targets, and as reagents for cell labeling and tracking.

Current diagnostic systems involve, for example, microarray technology,polymerase chain reaction (PCR), in situ hybridization, antibody-basedimmunoassays (e.g. enzyme-linked immunosorbant assays),chemiluminescence, nephelometry, and/or photometry. These systems cannotperform the diversity of assays at high sensitivity that is possiblewith an NMR-based nanosensor system.

Various non-NMR-based point of care bio-assays have been developed, suchas portable blood glucose meters that operate using test stripsimpregnated with glucose oxidase. However, these systems are generallynot as reliable as central hospital assays because they lack thesensitivity, calibration, and maintenance that a laboratory settingprovides. These portable systems also lack the sensitivity that ispossible with NMR-based nanosensor systems, and they cannot be easilyadapted for multiple analyte detection.

The above-cited Josephson et al. and Perez et al. documents describeapplication of classical NMR relaxation methods with nanosensors usingoff-the-shelf relaxometers and MRI units. However, these units requirelarge NMR RF coils and large magnets and are bulky, expensive, and arenot tailored for use with magnetic nanosensors.

There is a need for a less expensive, commercially-realizable NMR-basedanalyte detection device suitable for use with magnetic nanosensors.

SUMMARY OF THE INVENTION

The invention provides a small, integrated NMR-based analyte detectiondevice with superparamagnetic nanosensors which can be customized fordetection of any of a wide variety of analytes. The device may be used,for example, as a portable unit for point of care diagnosis and/or fielduse, or the device may be implanted for continuous or intermittentmonitoring of one or more biological species of interest in a patient.

In one configuration, the device contains an array of many small wells(e.g. 100, 1000, 10,000, or more “micro wells”) for containing a liquidsample, each well surrounded by a tiny radio frequency (RF) coil thatdetects an echo response produced by exposing the liquid sample in thewell to a bias magnetic field and RF excitation. The magnetic field iscreated using one or more magnets which may be part of the deviceitself, or may be external to the device. As used herein, “well” meansany localizer of a liquid sample, for example, an indentation, acontainer, a support, a channel, a reservoir, a sunken volume, acompartment, a recessed area, an enclosure with or without an opening, atube, a trough, a semipermeable membrane, an interface between twophases (e.g. an organic-inorganic interface, a hydrophilic-hydrophobicinterface, an oligophilic-oligophobic interface, and the like), and/oran interface between two fluids (gases and/or liquids).

Superparamagnetic nanoparticles are pre-deposited onto/into the microwells before introduction of the liquid sample, or, alternatively, thenanoparticles may be introduced into the wells along with the liquidsample. The nanoparticles have binding moieties on their surfaces, whichare operative to bind to (i) an analyte, (ii) another of the bindingmoieties, and/or (iii) an aggregation-inducing molecule in the liquidsample. These binding moieties may be customized such that aggregationor disaggregation of the nanoparticles occurs in the presence of one ormore analyte(s) to be detected.

The superparamagnetic character of the nanoparticles enhances water (orother solvent with free hydrogens) relaxation rates, an enhancement thatis altered by the aggregation or disaggregation of the particles. Thepresence and/or concentration(s) of the analyte(s) of interest can bedetected via NMR relaxation methods, even at extremely lowconcentrations, for example, 100 femto-molar and below. This increasedsensitivity can be achieved because of the effect of the analyte onaggregation, coupled with the effect of the state of aggregation on T2relaxation times.

In preferred embodiments, the devices offer a number of technologicaladvancements geared toward increasing sensitivity of analyte detection.These include, for example: (i) the use of a plurality of micro wells;(ii) the use of a well whose cross section varies spatially; (iii) thedesign of well/coil pairs with high filling factor; (iv) the positioningof an electrical element for echo signal conditioning in close proximityto the RF sensing coil; (v) the use of RF sensing coils with high Qfactor; (vi) the use of one or more rare earth magnets for producing thebias magnetic field; (vii) the positioning of the magnet(s) in closeproximity to the liquid sample; and (viii) the reduction in bandwidthmade possible by customization of the coated nanoparticles and well/coilgeometry for detection of a specific analyte. Embodiments of theinvention may make use of one or more of these technologicaladvancements in any combination.

The use of a plurality of micro wells further enhances detectionsensitivity, repeatability, and precision. Duplicate sampling wellsallow multiple, substantially simultaneous measurements of analyte(s).Furthermore, the binding moieties on the surfaces of the nanoparticlesused in the wells can be customized to provide greater sensitivity andprecision. For example, the concentration of nanoparticles and/orbinding moieties, and/or the types of binding moieties used in thedifferent wells can be varied, allowing for more sensitive detectionand/or more precise concentration measurement of the target analyte(s).Also, built-in self-calibration is enabled by the presence of one ormore wells reserved for calibration. For example, one or more wellshaving a known NMR relaxation characteristic that is substantiallyunaffected by the analyte can be dedicated for calibration.

In addition to the use of an array of well/coil pairs, anothertechnological feature improving analyte detection sensitivity of thedevices is the use of a well whose cross section varies spatially toconcentrate the analyte in the magnetic field. For example, each wellmay have a portion of larger cross-sectional area and a portion ofsmaller cross-sectional area. Superparamagentic nanoparticles coatedwith binding moieties differentially move analyte-containingaggregations in the intense magnetic field. A bias magnetic field movestarget analyte trapped in the aggregation of the magnetic nanoparticlesin the direction of the field from the large cross-section area of thewell into the small cross-section area of the well. In this way, theanalyte is concentrated in the small cross-sectional area of the well.The small cross-sectional area of the well is surrounded by an RF coilfor sensing the echo response of the solution. In this way, the analytemay be concentrated, for example, by a factor of about 1000, therebyincreasing sensitivity of the device about 1000 fold. The magnet(s)and/or magnetic field used to evoke an NMR relaxation response issynergistically used to concentrate the target analyte for improveddetection sensitivity. The device may include an array of many microwells and tiny RF coils surrounding the narrow portions of these wells.

Yet another technological feature improving analyte detectionsensitivity of the devices is the use of a well and RF coil configuredto provide a high filling factor. Filling factor, as used herein, is thevolume of liquid sample in a well divided by the volume circumscribed bythe RF coil. Improved analyte detection sensitivity can be achieved byusing a well and RF coil with a filling factor of at least about 0.1,preferably at least about 0.7, and more preferably about 1. For example,in one embodiment, the device contains an array of micro wellssurrounded by tiny RF coils, where each well/coil combination has afilling factor of about 1.

Still another technological feature improving analyte detectionsensitivity is the positioning of an electrical element for echo signalconditioning in close proximity to the RF coil. The small size of thewells facilitates placement of signal conditioning electronics within 1millimeter, for example, of the corresponding RF coil. The echo signalconditioning performed by the electrical element may include, forexample, amplification, rectification, and/or digitization of the echosignal. The electrical element (as the term is used herein in thesingular) may include one or more discrete electrical components.

A further technological feature improving analyte detection sensitivityis the use of RF sensing coils with high Q factor. Quality factor, or Qfactor, of an RF coil is a measure of its efficiency as an inductor, andis defined herein as the ratio of the inductive reactance of the RF coilto its resistance at a given frequency, for example, the Larmorfrequency. Using coils having high Q factor improves the sensitivity ofthe device.

Another technological feature enhancing analyte detection sensitivity isthe use of rare earth magnets to create the bias magnetic field.Examples of rare earth magnets include, for example, neodymium magnetssuch as Nd₂Fe₁₄13 (neodymium -iron-boron), and samarium cobalt magnetssuch as SmCO₅. This helps to maximize the strength of the magnetic fieldand improves sensitivity.

Another technological feature of the device is the positioning of themagnet(s), for example, rare earth magnet(s), used to produce the biasmagnetic field in close proximity to the liquid sample, for example,within 1 millimeter. This allows the generation of a bias magnetic fieldwith strength, for example, from about 1 to about 2 Tesla, as comparedwith commercial units that operate at 0.5 Tesla. The close proximity ofthe magnet to the liquid sample is facilitated by the micro design ofthe system and the integrated nature of the device.

Sensitivity of the device is also improved by the ability to use narrowbandwidth. Bandwidth in this sense is the amplitude roll off of thesignal processing chain. The wider the bandwidth, the flatter the rolloff with frequency. A wider bandwidth must be used when it is not clearwhat frequency is to be detected; however increased bandwidth results inincreased noise. Use of a narrower bandwidth results in less noise (andincreased signal-to-noise ratio, S/N), but may not be, possible unlessthe frequency to be detected is precisely known. The device makespossible the use of a reduced bandwidth, because the analyte to bedetected in each well is known and typically pre-determined, and thecoated nanoparticles and/or the well/coil geometry can be specificallycustomized for detection of the specific analyte. Multiple analytes maystill be detected, since different wells can be customized for detectionof different analytes, for example, by use of different binding moietieson the nanoparticles in the different wells.

Further customization of the electronics is possible. For example, theelectronics for a given well may be tuned to a specific, determinablefrequency characteristic based at least in part on the type ofanalyte/nanoparticle combination in the well and/or the concentration ofthe analyte and/or nanoparticle in the well. Furthermore, the use of oneor more pulse sequences may be developed for optimum detectionsensitivity/accuracy for a given analyte of interest, and/or for a givennanosensor.

In preferred embodiments, the device uses low power and is able tooperate in magnetic fields of less strength than current NMR systems,for example, less than about 7T, less than about 5T, less than about 4T,less than about 3T, less than about 2T, at about 1T, or less than about1T. In general, higher magnetic field strength could be used for assaysrequiring greater sensitivity, while lower magnetic field strength (forexample, below 1T) could be used for assays requiring less sensitivity.The power source may be any power source, for example, a battery or anyelectrical power source. An example power source would be a lithium ionbattery, such as (or similar to) a lithium ion battery used in cellulartelephones.

Aggregation of the nanoparticles is an equilibrium process.Nanoparticles may aggregate for a specific period of time (e.g.sufficient time for measurement to take place), then return to anonaggregated condition. Thus, the nanoparticles, localized in thewells, may be reused and would not need to be replaced following eachtest. This enhances the convenience and low cost of the unit.

Because of the adaptability of the nanoparticles (and binding moietieslinked thereto), the device may perform numerous bio-diagnosticfunctions. The device may be customized to perform a specific function,or adapted to perform more than one function, e.g. via changeablecartridges containing arrays of micro wells with customized, lyophilizednanoparticles deposited thereon.

The device may be used to perform bio-diagnostics rapidly, with highsensitivity, and at low cost. The device can be made portable and mayinclude a chip, module, or cartridge containing the sample wells, aswell as a handheld reader (remote or attached), making the unit usefulin the field by paramedics, emergency room personnel, or other medicalpersonnel for emergency medical care. Applications of the device includeuse by paramedics (e.g. in an ambulance or in the field), emergency roompersonnel, or other military or civilian medical personnel. The devicemay also be suitable for pediatric or adult home health care, forexample, for the monitoring of glucose levels in the treatment ofdiabetes. Home diagnostics may reduce the need for doctor and hospitalvisits. Implantable versions of the device may provide continuousmonitoring of species of interest, for example, glucose, coumadin,bacteria (e.g., post surgery), and/or drugs (e.g. for controlleddosing), to name a few.

The device may be used to detect a very wide range of biologicallyactive substances, as well as other analytes. Of current methods (e.g.chemiluminescence, nephelometry, photometry, and/or otheroptical/spectroscopic methods), no single approach can achieve thediversity of analysis that is possible with NMR, even without thesensitivity improvements made possible by embodiments described herein.The sensitivity improvements provided by embodiments of the inventiondescribed herein allow further breadth and adaptability of analysis overcurrent NMR techniques. For example, embodiments of the invention may beused or adapted for detection, for example, of any protein (e.g.,biomarkers for cancer, serum proteins, cell surface proteins, proteinfragments, modified proteins), any infectious disease (e.g., bacterialbased on surface or secreted molecules, virus based on core nucleicacids, cell surface modifications, and the like), as well as a widerange of gases and/or small molecules.

A wider range of drugs may be developed, due to the improved ability todetect and maintain appropriate dosages using the NMR device describedherein. Drugs may be administered either manually or automatically (e.g.via automatic drug metering equipment), and may be monitoredintermittently or continuously using the device. Dosage may therefore bemore accurately controlled, and drugs may be more accurately maintainedwithin therapeutic ranges, avoiding toxic concentrations in the body.Thus, drugs whose toxicity currently prevents their use may becomeapproved for therapeutic use when monitored with the device describedherein.

Medical conditions that may be rapidly diagnosed by the device forproper triaging and/or treatment include, for example, pain, fever,infection, cardiac conditions (e.g. stroke, thrombosis, and/or heartattack), gastrointestinal disorders, renal and urinary tract disorders,skin disorders, blood disorders, and/or cancers. Tests for infectiousdisease and cancer biomarkers for diseases not yet diagnosable bycurrent tests may be developed and performed using the NMR devicedescribed herein.

The device may be used for detection of chemical and/or biologicalweapons in the field, for example, nerve agents, blood agents, blisteragents, plumonary agents, incapacitating agents (e.g. lachrymatoryagents), anthrax, ebola, bubonic plague, cholera, tularemia,brucellosis, Q fever, typhus, encephalitis, smallpox, ricin, SEB,botulism toxin, saxitoxin, mycotoxin, and/or other toxins.

Because the devices are adaptable for detection of multiple analytes, aunit may be used to perform many ICU tests (including, e.g., PICU, SICU,NICU, CCU, and PACU) quickly and with a single blood draw. The tests mayalso be performed in the emergency room, in the physician's office, infield medicine (e.g. ambulances, military medical units, and the like),in the home, on the hospital floor, and/or in clinical labs. Themultiplexing capability of the devices also makes them a valuable toolin the drug discovery process, for example, by performing targetvalidation diagnostics.

Measurements for one or more analytes may be made, for example, based ona single draw, temporary draws, an intermittent feed, a semi-continuousfeed, a continuous feed, serial exposures, and/or continuous exposures.Measurements may include a detection of the presence of the one or moreanalytes and/or a measurement of the concentration of one or moreanalytes present in the sample.

Where the device is used as an implantable unit, one embodiment includesa semi-permeable pouch containing the nanoparticles and a set of biasfield permanent magnets. The implantable unit may be small, for example,about 2 mm diameter and about 5 mm long, and may be implanted in thearm. A reading may be made using a band, similar to a heart rate monitorband, that is placed around the arm such that the reader, outside thebody, is in proximity to the implant and measurements are performednon-invasively. The band may contain the RF coil and associatedelectronics.

In another embodiment of an implantable device, the unit may be a deepimplantable with RF excitation and/or sense coil(s), bias magnet(s),nanoparticle pouch, and power source all implanted. In anotherembodiment of an implantable device, the bias magnet(s), RF excitationand/or sense coil(s), and electronics are all external. The nanoparticlepouch is implanted, for example, in the arm, and a reader on a bandcontains the signal side bias magnet, RF coil(s), and electronics. Theband may be worn as would be a watch band, providing continuous orintermittent monitoring of an analyte of interest without wirespenetrating the body. The implant would not require a power source, thepower being provided by the reader worn externally by the patient.

In one aspect, the invention relates to a device for the detection of ananalyte, the device including: a support defining a well for holding aliquid sample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; and an RF coildisposed about the liquid sample, the RF coil being configured to detectan echo response produced by exposing the liquid sample to a biasmagnetic field created using one or more magnets and an RF excitation,wherein the RF coil has a characteristic dimension from about 10 μm toabout 1000 μm.

The characteristic dimension may be, for example, the diameter of thecoil (e.g. an inner diameter, an outer diameter, or an averagediameter), the length of the coil, or the depth of the coil. In certainembodiments, the RF coil has a diameter no greater than about 900 μm, nogreater than about 800 μm, no greater than about 700 μm, no greater thanabout 600 μm, no greater than about 500 μm, no greater than about 400μm, or no greater than about 300 μm. In certain embodiments, the RF coilhas a length or depth no greater than about 900 μm, no greater thanabout 800 μm, no greater than about 700 μm, no greater than about 600μm, no greater than about 500 μm, no greater than about 400 μm, or nogreater than about 300 μm.

In certain embodiments, the well and the RF coil are configured toprovide a filling factor of at least about 0.1, where the filling factoris the volume of the liquid sample in the well divided by the volumecircumscribed by the RF coil. In other embodiments, the filling factoris at least about 0.2, at least about 0.3, at least about 0.4, at leastabout 0.5, at least about 0.6, at least about 0.7, at least about 0.8,at least about 0.9, at least about 0.95, or about 1.

The well is preferably a micro well, meaning that the volume of theliquid sample in the well is less than about 1 mL. In certainembodiments, the volume of the liquid sample in the well is less thanabout 800 μL, less than about 700 μL, less than about 600 μL, less thanabout 500 μL, less than about 400 μL, less than about 300 μL, less thanabout 200 μL, less than about 100 μL, less than about 10 μL, less thanabout 1 μL, less than about 500 nL, less than about 300 nL, less thanabout 100 nL, less than about 50 nL, less than about 20 nL, less thanabout 5 nL, less than about 2 nL, or about 1 nL.

The RF coil is preferably a micro coil, meaning that the volumecircumscribed by the RF coil is less than about 1 mL. In certainembodiments, the volume circumscribed by the RF coil is less than about800 μL, less than about 700 μL, less than about 600 μL, less than about500 μL, less than about 400 μL, less than about 300 μL, less than about200 μL, less than about 100 μL, less than about 10 μL, less than about 1μL, less than about 500 nL, less than about 300 nL, less than about 100nL, less than about 50 nL, less than about 20 nL, less than about 5 nL,less than about 2 nL, or about 1 nL.

In certain embodiments either or both of (i) the volume of the liquidsample in the well and (ii) the volume circumscribed by the RF coilis/are less than about 1 mL. In certain embodiments, either or both of(i) the volume of the liquid sample in the well and (ii) the volumecircumscribed by the RF coil is/are less than about 800 μL, less thanabout 700 μL, less than about 600 μL, less than about 500 μL, less thanabout 400 μL, less than about 300 μL, less than about 200 μL, less thanabout 100A, less than about 10 μL, less than about 1 μL, less than about500 nL, less than about 300 nL, less than about 100 nL, less than about50 nL, less than about 20 nL, less than about 5 nL, less than about 2nL, or about 1 nL.

The device may further include an electrical element in communicationwith the RF coil, the electrical element configured to at leastpartially condition a signal corresponding to the echo response. Forexample, the electrical element may include a pre-amplifier, anamplifier, a rectifier, a transmitter, and/or a digitizer foramplifying, rectifying, transmitting, and/or digitizing the signalcorresponding to the echo response. In certain embodiments, theelectrical element is configured to do at least one of the following:(i) amplify the signal, (ii) rectify the signal, (iii) digitize thesignal. The electrical element (as the term is used herein in thesingular) may include one or more discrete electrical components. Forexample, the electrical element may include any combination of thecomponents shown in FIG. 14 such as the power splitter, power combiner,pre-amplifier, mixer, low-pass filter, and/or low noise amplifier.

The RF coil is preferably disposed sufficiently close to the electricalelement to provide a Q factor of at least 1, where the Q factor (qualityfactor) is the ratio of the inductive reactance of the RF coil to itsresistance at a given frequency, for example, the Larmor frequency. Incertain embodiments, the Q factor is at least about 5, at least about10, at least about 20, at least about 30, at least about 40, at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, at least about 100, or at least about 125. The proximityof the RF coil to the electrical element is important in thepreservation of the signal, allowing increased sensitivity.

The RF coil may be integrated with the support that defines the well,where the RF coil is disposed about the well. The support may be asubstrate, with the well etched from the substrate material.Alternatively, the support may form the base of the well, with the RFcoil itself serving as part or all of one or more sides of the well.

Preferably, the RF coil is disposed within one centimeter of theelectrical element. In certain embodiments, the RF coil is disposedwithin 5 millimeters of the electrical element, within 3 millimeters ofthe electrical element, within 2 millimeters of the electrical element,within 1 millimeter of the electrical element, within 500 micrometers ofthe electrical element, within 100 micrometers of the electricalelement, within 50 micrometers of the electrical element, or within 5micrometers of the electrical element.

The magnetic particles may include superparamagnetic nanoparticles withbinding moieties on their surfaces. The binding moieties are preferablyoperative to alter an aggregation of the magnetic particles as afunction of the presence or concentration of the analyte. The magneticparticles may include an oxide and/or a hydroxide of Fe, Si, Sn, An, Ti,Bi, Zr, and/or Zn. The magnetic particles are preferablysuperparamagnetic and have crystallite size from about 1 nm to about 100nm. The magnetic nanoparticles preferably have a metal oxide core ofabout 1 to about 25 nm, from about 3 to about 10 nm, or about 5 nm indiameter. The binding moieties may include one or more species of one ormore of the following: an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent, a metabolite of a therapeuticagent, a peptide, a polypeptide, a protein, a carbohydrate, apolysaccharide, a virus, and/or bacteria. For example, in oneembodiment, the binding moieties may include one, two, or more types ofoligonucleotides and/or one, two, or more types of proteins. The bindingmoieties may be a polymer, or may be part of a polymer that is linkedto, or otherwise associated with one or more of the magnetic particles.The binding moieties preferably include functional groups, for example,the binding moieties may include one or more species of one or more ofthe following: an amino group, a carboxyl group, a sulfhydryl group, anamine group, an imine group, an epoxy group, a hydroxyl group, a thiolgroup, an acrylate group, and/or an isocyano group.

The analyte may include one or more species of one or more of thefollowing: a protein, a peptide, a polypeptide, an amino acid, a nucleicacid, an oligonucleotide, a therapeutic agent, a metabolite of atherapeutic agent, RNA, DNA, an antibody, an organism, a virus,bacteria, a carbohydrate, a polysaccharide, and glucose. The analyte mayalso include, for example, a lipid, a gas (e.g., oxygen, carbondioxide), an electrolyte (e.g., sodium, potassium, chloride,bicarbonate, BUN, creatinine, glucose, magnesium, phosphate, calcium,ammonia, lactate), a lipoprotein, cholesterol, a fatty acid, aglycoprotein, a proteoglycan, and/or a lipopolysaccharide. Furthermore,as used herein, “detection of an analyte” may also mean measurement ofphysical properties of a solution containing one or more analytes, forexample, measurement of dipole moment, ionization,solubility/saturation, viscosity, gellation, crystallization, and/orphase changes of the solution.

The bias magnetic field may be substantially uniform, or it may have aspatial gradient. The device itself may include at least one of the oneor more magnets. At least one of the one or more magnets may be externalto the device. The RF excitation may be transmitted via an RF excitationcoil, separate from the RF coil disposed about the well (where the coildisposed about the well may be termed the “sensing” coil). In oneembodiment, the RF excitation may be transmitted via the RF coildisposed about the well. For example, the RF coil may both transmit theRF excitation and detect the echo response (the RF coil is both anexcitation coil and a sensing coil).

The device (or an element thereof) may be fabricated on a chip. Forexample, the device (or an element thereof) may be fabricated in a MEMS(micro electromechanical systems) process. The support (e.g., definingthe well) may include a plastic, polymer, film, fluid, fluid interface,liquid-liquid interface, organic (fluid) -inorganic (fluid) interface,and/or metals, for example. The support may include glass, Si, and/orSiGe. In certain embodiments, the liquid sample runs over the supportfor a continuous read (the liquid is not necessarily stationary on thesupport).

The RF coil may be deposited on a surface of the chip. The RF coil maybe a wound solenoid coil, a planar coil, a saddle coil, a Helmholtzcoil, or a MEMS solenoid coil.

In certain embodiments, the magnetic particles are deposited onto thesurface of the support defining the wells, for example, prior tointroduction of the liquid sample into the wells. The particles may bedeposited onto the support (e.g. a substrate) with a printer (e.g. amatrix dot printer or a laser printer), and/or the particles may bereconstitutable upon introduction of liquid. In certain embodiments, themagnetic particles are lyophilized.

The binding moieties are preferably operative to bind to at least one ofthe following (i, ii, and/or iii): (i) the analyte; (ii) another of thebinding moieties; and (iii) an aggregation-inducing molecule in theliquid sample. In this way, the binding moieties are operative toproduce an aggregate of multiply-linked magnetic particles as a functionof the presence or concentration of the analyte in the liquid sample. Anexample of an aggregation-inducing molecule is avidin and may be used,for example, where the binding moieties include biotin. In anotherembodiment, the aggregation-inducing molecule is biotin and the bindingmoieties include avidin. Alternatively, the aggregate of multiply-linkedmagnetic particles may be disaggregated as a function of the presence orconcentration of the analyte in the liquid sample. The bonds and/orlinks are preferably reversible, such that aggregation and/ordisaggregation is/are reversible, equilibrium-driven processes.

The aggregate may have an approximate size from about 100 nm to about500 nm in its largest dimension, for example. In certain embodiments,the aggregate has an approximate size greater than about 50 nm, greaterthan about 100 nm, greater than about 200 nm, or greater than about 300nm. The aggregate may contain, for example, from about 2 to about 20magnetic particles linked via the binding moieties. The magneticparticles may have an average size from about 5 nm to about 500 nm intheir largest dimension. In certain embodiments, the magnetic particleshave an average size less than about 500 nm in their largest dimension,less than about 200 nm in their largest dimension, less than about 100nm in their largest dimension, less than about 50 nm in their largestdimension, less than about 40 nm in their largest dimension, less thanabout 30 nm in their largest dimension, or less than about 20 nm intheir largest dimension. The largest dimension may be diameter, forexample.

The device may further include a reader configured to receive the signalcorresponding to the echo response. The reader may include an electricalelement for processing the signal and a display for indicating analytepresence or concentration. For example, the reader may determine achange in T2 relaxation time according to the signal corresponding tothe echo response, thereby indicating analyte presence or concentration.The reader may include a magnet for creation of the bias magnetic fieldand/or an RF excitation coil for providing the RF excitation. The readermay be spatially separated from the well and/or the sensing RF coil. Forexample, in the case where the device includes an implantable, thereader may be held outside the body. The device may be implantable andoperable without skin-penetrating wires. Other embodiments may includeone or more elements that penetrate the skin.

The device may be portable. For example, the device may weigh less thanabout 1 kilogram, less than about 500 grams, less than about 400 grams,or less than about 300 grams.

In another aspect, the invention relates to a device for the detectionof an analyte, the device including a plurality of wells for holding aliquid sample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto, and, for each of thewells: an RF coil disposed about the well, the RF coil configured todetect an echo response produced by exposing the liquid sample in thewell to a bias magnetic field created using one or more magnets and anRF excitation. The description of elements of the embodiments above canbe applied to this aspect of the invention as well.

The wells and the RF coils are preferably small. For example, in certainembodiments either or both of (i) the volume of the liquid sample ineach well and (ii) the volume circumscribed by each RF coil is/are lessthan about 1 mL. In certain embodiments, either or both of (i) thevolume of the liquid sample in each well and (ii) the volumecircumscribed by each RF coil is/are less than about 800 μL, less thanabout 700 μL, less than about 600 μL, less than about 500 μL, less thanabout 400 μL, less than about 300 μL, less than about 200 μL, less thanabout 100 μL, less than about 10 μL, less than about 1 μL, less thanabout 500 nL, less than about 300 nL, less than about 100 nL, less thanabout 50 nL, less than about 20 nL, less than about 5 nL, less thanabout 2 nL, or about 1 nL.

The wells are preferably arranged in an array, which may be, forexample, a 2-D or a 3-D array. The device may be configured to allowdistribution of liquid into the plurality of wells. For example,channels may be designed according to methods known in the art ofmicrofluidics to allow distribution of liquid into the plurality ofwells. For example, the design may enable pressure driven flow using oneor more positive displacements pumps or micropumps, such as syringepumps. The design may also or alternatively enable electrokinetic flowvia electroosmotic pumping.

The wells may include one or more wells dedicated for calibration. Forexample, one or more wells may have a known measurable characteristicthat is substantially unaffected by the analyte.

The plurality of wells may allow detection or concentration measurementof one or more analytes. For example, the magnetic particles havingdifferent binding moieties are disposed in different wells for detectionof multiple analytes. In certain embodiments, magnetic particles havingthe same binding moieties are disposed in different wells for replicatemeasurements, thereby improving accuracy (where improved accuracy maymean improved detection sensitivity). In certain embodiments, themagnetic particles having the same binding moieties (same species ofbinding moiety) are disposed in different wells for detection of varyinganalyte concentrations in the liquid sample. In certain embodiments, thedifferent wells have different concentrations of binding moietiesdisposed therein. In certain embodiments, the magnetic particles havingdifferent binding moieties are disposed in different wells for detectionof the analyte, where the different binding moieties promote aggregationor disaggregation of the magnetic particles in proportion toconcentration of the analyte.

The device may further include, for each of the wells, an electricalelement in communication with the RF coil corresponding to the well, theelectrical element configured to at least partially condition a signalcorresponding to the echo response. For example, each electrical elementmay include an amplifier, a rectifier, a transmitter, and/or a digitizerfor amplifying, rectifying, transmitting, and/or digitizing the signalcorresponding to the echo response. In certain embodiments, eachelectrical element is configured to do at least one of the following:(i) amplify the signal from the corresponding well, (ii) rectify thesignal, (iii) digitize the signal. The electrical element (as the termis used herein in the singular) may include one or more discreteelectrical components.

Each RF coil is preferably disposed sufficiently close to thecorresponding electrical element to provide a Q factor of at least 1,where the Q factor (quality factor) is the ratio of the inductivereactance of the RF coil to its resistance at a given frequency, forexample, the Larmor frequency. In certain embodiments, the Q factor isat least about 5, at least about 10, at least about 20, at least about30, at least about 40, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, at least about 100, orat least about 125.

The RF coils may be integrated with (e.g. embedded in) a substrate thatdefines the wells, where each RF coil is disposed about its respectivewell. Alternatively, a substrate may serve as the base of each of thewells, with each RF coil itself serving as part or all of one or moresides of the well. Preferably, each RF coil is disposed within onecentimeter of the corresponding electrical element. In certainembodiments, the RF coil is disposed within 5 millimeters of theelectrical element, within 3 millimeters of the electrical element,within 2 millimeters of the electrical element, within 1 millimeter ofthe electrical element, within 500 micrometers of the electricalelement, within 100 micrometers of the electrical element, within 50micrometers of the electrical element, or within 5 micrometers of theelectrical element.

The binding moieties are preferably operative to alter an aggregation ofthe magnetic particles as a function of the presence or concentration ofthe analyte. The magnetic particles may include superparamagneticnanoparticles with binding moieties on their surfaces. The magneticparticles may include an oxide and/or a hydroxide of Fe, Si, Sn, An, Ti,Bi, Zr, and/or Zn. The magnetic particles are preferablysuperparamagnetic and have crystallite size from about 1 nm to about 100nm. The magnetic nanoparticles preferably have a metal oxide core ofabout 1 to about 25 nm, from about 3 to about 10 nm, or about 5 nm indiameter. The binding moieties may include one or more species of one ormore of the following: an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent, a metabolite of a therapeuticagent, a peptide, a polypeptide, a protein, a carbohydrate, apolysaccharide, a virus, and/or bacteria. For example, in oneembodiment, the binding moieties may include one, two, or more types ofoligonucleotides and/or one, two, or more types of proteins. The bindingmoieties may be a polymer, or may be part of a polymer that is linkedto, or otherwise associated with one or more of the magnetic particles.The binding moieties preferably include functional groups, for example,the binding moieties may include one or more species of one or more ofthe following: an amino group, a carboxyl group, a sulfhydryl group, anamine group, an imine group, an epoxy group, a hydroxyl group, a thiolgroup, an acrylate group, and/or an isocyano group.

The analyte may include one or more species of one or more of thefollowing: a small organic molecule, a protein, a peptide, apolypeptide, an amino acid, a nucleic acid, an oligonucleotide, atherapeutic agent, a metabolite of a therapeutic agent, RNA, DNA, anantibody, an organism, a virus, bacteria, a carbohydrate, apolysaccharide, and glucose. The analyte may also include, for example,a lipid, a gas (e.g., oxygen, carbon dioxide), an electrolyte (e.g.,sodium, potassium, calcium, ammonia, lactate, lactic acid), alipoprotein, cholesterol, a fatty acid, a glycoprotein, a proteoglycan,and/or a lipopolysaccharide. Furthermore, “detection of an analyte” mayalso mean measurement of physical properties of a solution containingone or more analytes, for example, measurement of dipole moment,ionization, solubility/saturation, viscosity, gellation,crystallization, and/or phase changes of the solution.

The bias magnetic field may be substantially uniform, or it may have aspatial gradient. The device itself may include at least one of the oneor more magnets. At least one of the one or more magnets may be externalto the device. The RF excitation may be transmitted via an RF excitationcoil, separate from the RF coils disposed about the wells (where thecoils disposed about the wells may be termed the “sensing” coils). Inone embodiment, the RF excitation may be transmitted via the RF coilsdisposed about the wells. For example, the RF coils may both transmitthe RF excitation and detect the echo responses from the liquid samplesin their respective wells (where each of the RF coils acts as both anexcitation coil and a sensing coil).

The device (or an element thereof) may be fabricated on a chip. Forexample, the device (or an element thereof) may be fabricated in a MEMS(micro electromechanical systems) process.

The RF coils may be deposited on a surface of the chip. The RF coils mayinclude wound solenoid coils, planar coils, saddle coils, Helmholtzcoils, and/or MEMS solenoid coils.

In certain embodiments, the magnetic particles are deposited ontosurfaces of the wells (e.g. a substrate from which the wells are etchedor built up), for example, prior to introduction of the liquid sampleinto the wells. The particles may be deposited onto the surfaces with aprinter, and/or the particles may be reconstitutable upon introductionof liquid. In certain embodiments, the magnetic particles arelyophilized.

The binding moieties are preferably operative to bind to at least one ofthe following (i, ii, and/or iii): (i) the analyte; (ii) another of thebinding moieties; and (iii) an aggregation-inducing molecule in theliquid sample. In this way, the binding moieties are operative toproduce an aggregate of multiply-linked magnetic particles as a functionof the presence or concentration of the analyte in the liquid sample. Anexample of an aggregation-inducing molecule is avidin and may be used,for example, where the binding moieties include biotin. In anotherembodiment, the aggregation-inducing molecule is biotin and the bindingmoieties include avidin. Alternatively, the aggregate of multiply-linkedmagnetic particles may be disaggregated as a function of the presence orconcentration of the analyte in the liquid sample.

The device may include a replaceable and/or interchangeable cartridgecontaining the array of wells pre-loaded with dried (e.g. lyophilized)magnetic particles. The cartridge may be designed for detection and/orconcentration measurement of a particular analyte. The device may beusable with different cartridges, each designed for detection and/orconcentration measurements of different analytes. The cartridge may besized for convenient insertion into and ejection from a housingcontaining one or more of the magnets and/or an RF excitation coil.

The device may further include a reader configured to receive thesignals corresponding to the echo responses from the wells. The readermay include an electrical element for processing the signals and adisplay for indicating analyte presence or concentration. For example,the reader may determine a change in T2 relaxation time according to thesignals corresponding to the echo responses, thereby indicating analytepresence or concentration. The reader may include a magnet for creationof the bias magnetic field and/or an RF excitation coil for providingthe RF excitation. The reader may be spatially separated from the wellsand/or the sensing RF coil. For example, in the case where the device isadapted for implementation into a mammal, the reader may be held outsidethe body. The device may be implantable and operable withoutskin-penetrating wires. Other embodiments may include one or moreelements that penetrate the skin.

The device may be portable. For example, the device may weigh less thanabout 1 kilogram, less than about 500 grams, less than about 400 grams,or less than about 300 grams.

In yet another aspect, the invention relates to a device including asupport defining one or more wells for holding a liquid sample; anddisposed on the support, for reconstitution within the one or morewells, dried superparamagnetic particles having binding moieties linkedthereto, where the binding moieties are operative to alter anaggregation of the magnetic particles in the liquid sample as a functionof the presence or concentration of an analyte in the liquid sample. Thedescription of elements of the embodiments above can be applied to thisaspect of the invention as well.

In one embodiment, the device further includes, for each of the one ormore wells, an RF coil disposed about the well and an electrical elementin communication with the RF coil, wherein the RF coil is configured todetect an echo response produced by exposing the liquid sample in thewell to a bias magnetic field created using one or more magnets and anRF excitation, wherein the electrical element is configured to at leastpartially condition a signal corresponding to the echo response.

In certain embodiments, the device is a component of an analytedetection system. For example, in certain embodiments, the device is areplaceable and/or interchangeable cartridge containing the array ofwells pre-loaded with dried (e.g. lyophilized) magnetic particles. Thecartridge may be designed for detection and/or concentration measurementof a particular analyte. Different cartridges may be designed fordetection and/or concentration measurements of different analytes. Thecartridges may themselves include the RF coils configured to detect echoresponses from the liquid samples in corresponding wells, or the RFcoils may be separate from the cartridges. The cartridges may bedesigned for operation with a console, for example, where the consoleincludes one or more magnets for producing the bias magnetic fieldand/or an RF excitation coil for transmitting the RF excitation.

In certain embodiments, the device further includes an RF excitationcoil for transmitting the RF excitation, where the RF excitation coil isseparate from the one or more RF coils disposed about the one or morewells (e.g. the RF coils for sensing echo response). For each of the oneor more wells, the respective RF coil is disposed within one centimeter,within one millimeter, or within 100 μm of the electrical element incommunication with the RF coil. The electrical element may be configuredto do at least one of the following: (i) amplify the signalcorresponding to the echo response; (ii) rectify the signal; (iii)digitize the signal.

The binding moieties are preferably operative to alter an aggregation ofthe superparamagnetic particles as a function of the presence orconcentration of the analyte. The superparamagnetic particles mayinclude superparamagnetic nanoparticles with binding moieties on theirsurfaces. The superparamagnetic particles may include an oxide and/or ahydroxide of Fe, Si, Sn, An, Ti, Bi, Zr, and/or Zn. Thesuperparamagnetic particles preferably have crystallite size from about1 nm to about 100 nm. The superparamagnetic particles preferably have ametal oxide core of about 1 to about 25 nm, from about 3 to about 10 nm,or about 5 nm in diameter. The binding moieties may include one or morespecies of one or more of the following: an amino acid, a nucleic acid,an oligonucleotide, a therapeutic agent, a metabolite of a therapeuticagent, a peptide, a polypeptide, a protein, a carbohydrate, apolysaccharide, a virus, and/or bacteria. For example, in oneembodiment, the binding moieties may include one, two, or more types ofoligonucleotides and/or one, two, or more types of proteins. The bindingmoieties may be a polymer, or may be part of a polymer that is linkedto, or otherwise associated with one or more of the superparamagneticparticles. The binding moieties preferably include functional groups,for example, the binding moieties may include one or more species of oneor more of the following: an amino group, a carboxyl group, a sulfhydrylgroup, an amine group, an imine group, an epoxy group, a hydroxyl group,a thiol group, an acrylate group, and/or an isocyano group.

The analyte may include one or more species of one or more of thefollowing: a protein, a peptide, a polypeptide, an amino acid, a nucleicacid, an oligonucleotide, a therapeutic agent, a metabolite of atherapeutic agent, RNA, DNA, an antibody, an organism, a virus,bacteria, a carbohydrate, a polysaccharide, and glucose. The analyte mayalso include, for example, a lipid, a gas (e.g., oxygen, carbondioxide), an electrolyte (e.g., sodium, potassium, calcium, ammonia,lactate, lactic acid), a lipoprotein, cholesterol, a fatty acid, aglycoprotein, a proteoglycan, and/or a lipopolysaccharide. Furthermore,“detection of an analyte” may also mean measurement of physicalproperties of a solution containing one or more analytes, for example,measurement of dipole moment, ionization, solubility/saturation,viscosity, gellation, crystallization, and/or phase changes of thesolution.

For each of the one or more wells, the well and the RF coil disposedabout the well are preferably configured to provide a filling factor ofat least about 0.7, at least about 0.9, or about 1.

The device may further include a reader configured to receive, for eachof the wells, the signal corresponding to the echo response from therespective well.

In another aspect of the invention, in invention relates to a deviceincluding a support defining one or more wells for holding a liquidsample, the sample comprising magnetic particles and an analyte, themagnetic particles having binding moieties linked thereto, wherein thebinding moieties are operative to alter an aggregation of said magneticparticles in the liquid sample as a function of the presence orconcentration of the analyte in the liquid sample, and wherein at leastone of the wells has a varying cross section such that, in the presenceof a magnetic field, aggregations of the particles move from an area oflarger cross section to an area of smaller cross section, therebyconcentrating the analyte carried with the aggregations. The descriptionof elements of the embodiments above can be applied to this aspect ofthe invention as well.

In certain embodiments, the device includes, for each of the one or morewells, an RF coil disposed about the well and an electrical element incommunication with the RF coil, wherein the RF coil is configured todetect an echo response produced by exposing the liquid sample in thewell to a bias magnetic field created using one or more magnets and anRF excitation, wherein the electrical element is configured to at leastpartially condition a signal corresponding to the echo response. Inpreferred embodiments, at least one of the binding moieties is operativeto bind to at least one of the following (thereby producing theaggregations): (i) the analyte; (ii) another of the binding moieties;(iii) an aggregation-inducing molecule in the liquid sample.

The binding moieties may include one or more species of one or more ofthe following: an amino acid, a nucleic acid, an oligonucleotide, atherapeutic agent, a metabolite of a therapeutic agent, a peptide, apolypeptide, a protein, a carbohydrate, a polysaccharide, a virus,and/or bacteria.

The analyte may include one or more species of one or more of thefollowing: a protein, a peptide, a polypeptide, an amino acid, a nucleicacid, an oligonucleotide, a therapeutic agent, a metabolite of atherapeutic agent, RNA, DNA, an antibody, an organism, a virus,bacteria, a carbohydrate, a polysaccharide, and glucose. The analyte mayalso include, for example, a lipid, a gas (e.g., oxygen, carbondioxide), an electrolyte (e.g., sodium, potassium, calcium, ammonia,lactate, lactic acid), a lipoprotein, cholesterol, a fatty acid, aglycoprotein, a proteoglycan, and/or a lipopolysaccharide. Furthermore,“detection of an analyte” may also mean measurement of physicalproperties of a solution containing one or more analytes, for example,measurement of dipole moment, ionization, solubility/saturation,viscosity, gellation, crystallization, and/or phase changes of thesolution.

In another aspect, the invention relates to a device for detection of ananalyte, the device including a support defining a well for holding aliquid sample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; and an RF coildisposed about the liquid sample, the RF coil configured to detect anecho response produced by exposing the liquid sample to a bias magneticfield created using one or more magnets and an RF excitation, the welland the RF coil configured to provide a filling factor of at least about0.1. In certain embodiments, the filling factor is at least about 0.7,at least about 0.9, at least about 0.95, or about 1. The description ofelements of the embodiments above can be applied to this aspect of theinvention as well.

In another aspect, the invention relates to a device for detection of ananalyte, the device including a support defining a well for holding aliquid sample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; and an RF coildisposed about the liquid sample, the RF coil configured to detect anecho response produced by exposing the liquid sample to a bias magneticfield created using one or more magnets and an RF excitation, wherein atleast one of the following is less than about 1 mL: (i) the volumecircumscribed by the RF coil; (ii) the volume of the liquid sample. Incertain embodiments either or both of (i) and (ii) is/are less thanabout 800 μL, less than about 700 μL, less than about 600 μL, less thanabout 500 μL, less than about 400 μL, less than about 300 μL, less thanabout 200 μL, less than about 100 μL, less than about 10 μL, less thanabout 1 μL, less than about 500 nL, less than about 300 nL, less thanabout 100 nL, less than about 50 nL, less than about 20 nL, less thanabout 5 nL, less than about 2 nL, or about 1 nL. The description ofelements of the embodiments above can be applied to this aspect of theinvention as well.

In another aspect, the invention relates to a device for detection of ananalyte, the device including a support defining a well for holding aliquid sample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; an RF coil disposedabout the liquid sample, the RF coil configured to detect an echoresponse produced by exposing the liquid sample to a bias magnetic fieldcreated using one or more magnets and an RF excitation; and anelectrical element in communication with the RF coil, the electricalelement configured to at least partially condition a signalcorresponding to the echo response, wherein the RF coil is disposedsufficiently close to the electrical element to provide a Q factor of atleast 1, where the Q factor (quality factor) is the ratio of theinductive reactance of the RF coil to its resistance at a givenfrequency, for example, the Larmor frequency. In certain embodiments,the Q factor is at least about 5, at least about 10, at least about 20,at least about 30, at least about 40, at least about 50, at least about60, at least about 70, at least about 80, at least about 90, at leastabout 100, or at least about 125. The description of elements of theembodiments above can be applied to this aspect of the invention aswell.

The RF coil may be integrated with the support that defines the well,where the RF coil is disposed about the well. The support may be asubstrate, with the well etched from the substrate material.Alternatively, the support may form the base of the well, with the RFcoil itself serving as part or all of one or more sides of the well.Preferably, the RF coil is disposed within one centimeter of theelectrical element. In certain embodiments, the RF coil is disposedwithin 5 millimeters of the electrical element, within 3 millimeters ofthe electrical element, within 2 millimeters of the electrical element,within 1 millimeter of the electrical element, within 500 micrometers ofthe electrical element, within 100 micrometers of the electricalelement, within 50 micrometers of the electrical element, or within 5micrometers of the electrical element.

In another aspect, the invention relates to a method of measuring one ormore analytes in a sample using any one of (or any combination of) thefollowing devices:

(i) a device including: a support defining a well for holding a liquidsample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; and an RF coildisposed about the liquid sample, the RF coil configured to detect anecho response produced by exposing the liquid sample to a bias magneticfield created using one or more magnets and an RF excitation, whereinthe RF coil has a characteristic dimension from about 10 μm to about1000 μm;(ii) a device including a plurality of wells for holding a liquid sampleincluding magnetic particles and the analyte, the magnetic particleshaving binding moieties linked thereto, and, for each of the wells: anRF coil disposed about the well, the RF coil configured to detect anecho response produced by exposing the liquid sample in the well to abias magnetic field created using one or more magnets and an RFexcitation;(iii) a device including a support defining one or more wells forholding a liquid sample; and disposed on the support, for reconstitutionwithin the one or more wells, dried superparamagnetic particles havingbinding moieties linked thereto, where the binding moieties areoperative to alter an aggregation of the magnetic particles in theliquid sample as a function of the presence or concentration of ananalyte in the liquid sample;(iv) a device including a support defining one or more wells for holdinga liquid sample, the sample comprising magnetic particles and ananalyte, the magnetic particles having binding moieties linked thereto,wherein the binding moieties are operative to alter an aggregation ofsaid magnetic particles in the liquid sample as a function of thepresence or concentration of the analyte in the liquid sample, andwherein at least one of the wells has a varying cross section such that,in the presence of a magnetic field, aggregations of the particles movefrom an area of larger cross section to an area of smaller crosssection, thereby concentrating the analyte carried with theaggregations;(v) a device including a support defining a well for holding a liquidsample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; and an RF coildisposed about the liquid sample, the RF coil configured to detect anecho response produced by exposing the liquid sample to a bias magneticfield created using one or more magnets and an RF excitation, the welland the RF coil configured to provide a filling factor of at least about0.1;(vi) a device including a support defining a well for holding a liquidsample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; and an RF coildisposed about the liquid sample, the RF coil configured to detect anecho response produced by exposing the liquid sample to a bias magneticfield created using one or more magnets and an RF excitation, wherein atleast one of the following is less than about 1 mL: (A) the volumecircumscribed by the RF coil; (B) the volume of the liquid sample;and/or(vii) a device including a support defining a well for holding a liquidsample including magnetic particles and the analyte, the magneticparticles having binding moieties linked thereto; an RF coil disposedabout the liquid sample, the RF coil configured to detect an echoresponse produced by exposing the liquid sample to a bias magnetic fieldcreated using one or more magnets and an RF excitation; and anelectrical element in communication with the RF coil, the electricalelement configured to at least partially condition a signalcorresponding to the echo response, wherein the RF coil is disposedsufficiently close to the electrical element to provide a Q factor of atleast 1, where the Q factor (quality factor) is the ratio of theinductive reactance of the RF coil to its resistance at a givenfrequency, for example, the Larmor frequency. The description ofelements of the embodiments above can be applied to this aspect of theinvention as well.

In certain embodiments, the one or more analytes measured by thedevice(s) include one or more biologically active substances. In certainembodiments, the sample includes a research sample, a cell sample,and/or an organism-derived sample. In certain embodiments, the method isperformed in vivo (for example, where the device is implantable). Incertain embodiments, the measuring step includes determining theconcentration of the one or more analytes in the sample. In certainembodiments, the measuring step includes detecting the presence of theone or more analytes in the sample. In certain embodiments, themeasuring step includes continuously monitoring the one or moreanalytes, semi-continuously monitoring the one or more analytes, and/orintermittently monitoring the one or more analytes. In certainembodiments, the measuring step includes continuously monitoring the oneor more analytes in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention can be better understood withreference to the drawings described below, and the claims. The drawingsare not necessarily to scale, emphasis instead generally being placedupon illustrating the principles of the invention. In the drawings, likenumerals are used to indicate like parts throughout the various views.

While the invention is particularly shown and described herein withreference to specific examples and specific embodiments, it should beunderstood by those skilled in the art that various changes in form anddetail may be made therein without departing from the spirit and scopeof the invention.

FIG. 1 is a schematic diagram of an NMR system for detection of an echoresponse of a sample to an RF excitation, according to an illustrativeembodiment of the invention.

FIGS. 2A-2E illustrate micro NMR coil (RF coil) designs including awound solenoid coil (FIG. 2A), a planar coil (FIG. 2B), a MEMS solenoidcoil (FIG. 2C), a MEMS Helmholz coil (FIG. 2D), and a saddle coil (FIG.2E), according to an illustrative embodiment of the invention.

FIG. 3 is a schematic diagram of an NMR system employing magneticnanoparticles in a micro well for holding a liquid sample, the wellsurrounded by an RF coil on a substrate (chip), where the magnet forcreating the bias magnetic field lies on the substrate, according to anillustrative embodiment of the invention.

FIG. 4A is a schematic diagram of an NMR system employing magneticnanoparticles in a micro well, where the magnet for creating atop-to-bottom bias magnetic field does not lie on the chip (the magnetis above and below the well), according to an illustrative embodiment ofthe invention.

FIG. 4B is a schematic diagram of an NMR system employing magneticnanoparticles in a micro well, where the magnet for creating aside-to-side bias magnetic field does not lie on the chip (the magnet isadjacent to the well), according to an illustrative embodiment of theinvention.

FIG. 5A is a schematic diagram of an NMR system including a single wellwith external RF excitation coil and external bias magnet, according toan illustrative embodiment of the invention.

FIG. 5B is a schematic diagram of an NMR system including an array ofwells with external RF excitation coil and external bias magnet,according to an illustrative embodiment of the invention.

FIG. 6A is a schematic diagram of an NMR system including a single well,according to an illustrative embodiment of the invention.

FIG. 6B is a schematic diagram of an NMR system including amultiple-well array, according to an illustrative embodiment of theinvention.

FIG. 6C is a schematic diagram of an NMR system including multiple wellscontaining different nanoparticles for detection of different analytes,according to an illustrative embodiment of the invention.

FIG. 6D is a schematic diagram of an NMR system including groups ofwells with identical nanoparticles for obtaining multiple data points(redundant measurements) for increased precision, sensitivity, and/orrepeatability, according to an illustrative embodiment of the invention.

FIG. 7 is a block diagram depicting basic components of an NMR system,including electrical components, according to an illustrative embodimentof the invention.

FIG. 8 is a block diagram of an NMR system including multiple wells andsensing coils and an external RF excitation coil, according to anillustrative embodiment of the invention.

FIG. 9 is a block diagram of an NMR system including multiple wells andsensing coils without an external RF excitation coil (the sensing coilsalso serve as excitation coils), according to an illustrative embodimentof the invention.

FIG. 10 is a schematic diagram of a chip module receiver/reader,according to an illustrative embodiment of the invention.

FIG. 11 is a schematic diagram of a magnetic analyte concentrator,according to an illustrative embodiment of the invention.

FIG. 12 is a schematic diagram of a syringe analyte concentrator,according to an illustrative embodiment of the invention.

FIG. 13 is a schematic diagram of a membrane analyte concentrator,according to an illustrative embodiment of the invention.

FIG. 14 is a schematic diagram of an electronics set-up for NMRmeasurement, according to an illustrative embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It is contemplated that devices, systems, methods, and processes of theclaimed invention encompass variations and adaptations developed usinginformation from the embodiments described herein. Adaptation and/ormodification of the devices, systems, methods, and processes describedherein may be performed by those of ordinary skill in the relevant art.

Throughout the description, where devices and systems are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are devices andsystems of the present invention that consist essentially of, or consistof, the recited components, and that there are processes and methodsaccording to the present invention that consist essentially of, orconsist of, the recited processing steps. Use of the term “about” withrespect to any quantity is contemplated to include that quantity. Forexample, “about 10 μm” is contemplated herein to include “10 μm”, aswell as values understood in the art to be approximately 10 μm withrespect to the entity described.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim.

As used herein, “nanoparticle” is understood to mean a particle havingat least one dimension less than about 200 nm.

As used herein, “microparticle” is understood to mean a particle havingat least one dimension less than about 200 μm.

As used herein, “characteristic dimension” of an entity is a dimensionthat is characteristic of the entity; for example, height is acharacteristic dimension of a human being.

As used herein, “filling factor” is understood to mean the volume of theliquid sample in a well divided by the volume circumscribed by the RFcoil.

As used herein, “quality factor” or “Q factor” of an RF coil isunderstood to be a measure of its efficiency as an inductor, and isdefined as the ratio of the inductive reactance of the RF coil to itsresistance at a given frequency, for example, the Larmor frequency.

As used herein, “linked” is understood to mean attached or bound bycovalent bonds, non-covalent bonds, and/or linked via Van der Waalsforces, hydrogen bonds, and/or other intermolecular forces.

The following headers are provided as a general organizational guide anddo not serve to limit support for any given element of the invention toa particular section of the Description.

Nanoparticles

The nanoparticles described herein include those described in U.S.Patent Application Publication No. US 2003/0092029, the text of which isincorporated herein by reference. The nanoparticles may be in the formof conjugates, that is, a magnetic nanoparticle with one or more bindingmoieties (e.g. an oligonucleotide, nucleic acid, polypeptide, orpolysaccharide) linked thereto. The binding moiety causes a specificinteraction with a target analyte (or an aggregation-inducing molecule,such as avidin). The binding moiety specifically binds to a selectedtarget analyte, for example, a nucleic acid, polypeptide, orpolysaccharide, or the binding moiety can be designed to bind to anotherbinding moiety to form an aggregate that is cleaved by the targetmolecule. Binding causes aggregation of the conjugates, resulting in adecrease of the spin-spin relaxation time (T2) of adjacent water protonsin an aqueous solution (or free protons in a non-aqueous solvent).Cleavage causes dispersal of the aggregate into separate conjugates,resulting in an increase of the spin-spin relaxation time (T2) ofadjacent water protons in an aqueous solution (or free protons in anon-aqueous solvent).

The conjugates have high relaxivity owing to the superparamagnetism oftheir iron or metal oxide. The conjugates have an R1 relaxivity fromabout 5 to about 30 mM⁻¹sec⁻¹, e.g., 10, 15, 20, or 25 mM⁻¹sec⁻¹. Theconjugates have an R2 relaxivity between about 15 and 100 mM⁻¹sec⁻¹,e.g., 25, 50, 75, or 90 mM⁻¹sec⁻¹. The conjugates generally have a ratioof R2 to R1 from about 1.5 to about 4, e.g., about 2, 2.5, or 3. Theconjugates generally have an iron oxide content that is greater thanabout 10% of the total mass of the particle, e.g., greater than 15, 20,25 or 30 percent.

The nanoparticles can be monodisperse (a single crystal of a magneticmaterial, e.g., metal oxide, such as superparamagnetic iron oxide, pernanoparticle) or polydisperse (a plurality of crystals, e.g., 2, 3, or4, per nanoparticle). The magnetic metal oxide can also comprise cobalt,magnesium, zinc, or mixtures of these metals with iron. The term“magnetic” as used herein means materials of high positive magneticsusceptibility such as paramagnetic compounds, superparamagneticcompounds, and magnetite, gamma ferric oxide, or metallic iron.Important features and elements of nanoparticles that are useful toproduce conjugates include: (i) a high relaxivity, i.e., strong effecton water (or other solvent) relaxation, (ii) a functional group to whichthe binding moiety can be covalently attached, (iii) a low non-specificbinding of interactive moieties to the nanoparticle, and/or (iv)stability in solution, i.e., the nanoparticles do not precipitate.

The nanoparticles may be linked to the binding moieties via functionalgroups. In some embodiments, the nanoparticles are associated with apolymer that includes the functional groups, and that also serves tokeep the metal oxides dispersed from each other. The polymer can be asynthetic polymer, such as, but not limited to, polyethylene glycol orsilane, natural polymers, or derivatives of either synthetic or naturalpolymers or a combination of these. The polymer may be hydrophilic. Insome embodiments, the polymer “coating” is not a continuous film aroundthe magnetic metal oxide, but is a “mesh” or “cloud” of extended polymerchains attached to and surrounding the metal oxide. The polymer cancomprise polysaccharides and derivatives, including dextran, pullanan,carboxydextran, carboxmethyl dextran, and/or reduced carboxymethyldextran. The metal oxide can be a collection of one or more crystalsthat contact each other, or that are individually entrapped orsurrounded by the polymer.

In other embodiments, the nanoparticles are associated withnon-polymeric functional group compositions. Methods of synthesizingstabilized, functionalized nanoparticles without associated polymers aredescribed, for example, in Halbreich et al., Biochimie, 80 (5-6):379-90,1998.

The nanoparticles may have an overall size of less than about 1-100 nm.The metal oxides may be in the form of crystals about 1-25 nm, e.g.,about 3-10 nm, or about 5 nm in diameter. The polymer component in someembodiments can be in the form of a coating, e.g., about 5 to 20 nmthick or more. The overall size of the nanoparticles is about 15 to 200nm, e.g., about 20 to 100 nm, about 40 to 60 nm; or about 50 nm.

The nanoparticles may be prepared in a variety of ways. It is preferredthat the nanoparticle have functional groups that link the nanoparticleto the binding moiety.

Carboxy functionalized nanoparticles can be made, for example, accordingto the method of Gorman (see WO 00/61191). In this method, reducedcarboxymethyl (CM) dextran is synthesized from commercial dextran. TheCM-dextran and iron salts are mixed together and are then neutralizedwith ammonium hydroxide. The resulting carboxy functionalizednanoparticles can be used for coupling amino functionalizedoligonucleotides.

Carboxy-functionalized nanoparticles can also be made frompolysaccharide coated nanoparticles by reaction with bromo orchloroacetic acid in strong base to attach carboxyl groups. In addition,carboxy-functionalized particles can be made from amino-functionalizednanoparticles by converting amino to carboxy groups by the use ofreagents such as succinic anhydride or maleic anhydride.

Nanoparticle size can be controlled by adjusting reaction conditions,for example, by using low temperature during the neutralization of ironsalts with a base as described in U.S. Pat. No. 5,262,176. Uniformparticle size materials can also be made by fractionating the particlesusing centrifugation, ultrafiltration, or gel filtration, as described,for example in U.S. Pat. No. 5,492,814.

Nanoparticles can also be synthesized according to the method of Molday(Molday, R. S, and D. MacKenzie, “Immunospecific ferromagneticiron-dextran reagents for the labeling and magnetic separation ofcells,” J. Immunol. Methods, 1982, 52(3)353-67, and treated withperiodate to form aldehyde groups. The aldehyde-containing nanoparticlescan then be reacted with a diamine (e.g., ethylene diamine orhexanediamine), which will form a Schiff base, followed by reductionwith sodium borohydride or sodium cyanoborohydride.

Dextran-coated nanoparticles can be made and cross-linked withepichlorohydrin. The addition of ammonia reacts with epoxy groups togenerate amine groups, see Hogemann, D., et al., Improvement of MRIprobes to allow efficient detection of gene expression Bioconjug. Chem.2000, 11(6):941-6, and Josephson et al., “High-efficiency intracellularmagnetic labeling with novel superparamagnetic-Tat peptide conjugates,”Bioconjug. Chem., 1999, 10(2):186-91. This material is known ascross-linked iron oxide or “CLIO” and when functionalized with amine isreferred to as amine-CLIO or NH₂—CLIO.

Carboxy-functionalized nanoparticles can be converted toamino-functionalized magnetic particles by the use of water-solublecarbodiimides and diamines such as ethylene diamine or hexane diamine.

Avidin or streptavidin can be attached to nanoparticles for use with abiotinylated binding moiety, such as an oligonucleotide or polypeptide.See, e.g., Shen et al., “Magnetically labeled secretin retains receptoraffinity to pancreas acinar cells,” Bioconjug. Chem., 1996, 7(3):311-6.Similarly, biotin can be attached to a nanoparticle for use with anavidin-labeled binding moiety.

Low molecular weight compounds can be separated from the nanoparticlesby ultra-filtration, dialysis, magnetic separation, or other means. Theunreacted oligonucleotides can be separated from theoligonucleotide-nanoparticle conjugates, e.g., by magnetic separation orsize exclusion chromatography.

Binding Moieties

In general, a binding moiety is a molecule, synthetic or natural, thatspecifically binds or otherwise links to, e.g., covalently ornon-covalently binds to or hybridizes with, a target molecule, or withanother binding moiety (or, in certain embodiments, with an aggregationinducing molecule). For example, the binding moiety can be a syntheticoligonucleotide that hybridizes to a specific complementary nucleic acidtarget. The binding moiety can also be an antibody directed toward anantigen or any protein-protein interaction. Also, the binding moiety canbe a polysaccharide that binds to a corresponding target. In certainembodiments, the binding moieties can be designed or selected to serve,when bound to another binding moiety, as substrates for a targetmolecule such as enzyme in solution.

Binding moieties include, for example, oligonucleotide binding moieties,polypeptide binding moieties, antibody binding moieties, andpolysaccharide binding moieties.

Oligonucleotide Binding Moieties

In certain embodiments, the binding moieties are oligonucleotides,attached/linked to the nanoparticles using any of a variety ofchemistries, by a single, e.g., covalent, bond, e.g., at the 3′ or 5′end to a functional group on the nanoparticle.

An oligonucleotide binding moiety can be constructed using chemicalsynthesis. A double-stranded DNA binding moiety can be constructed byenzymatic ligation reactions using procedures known in the art. Forexample, a nucleic acid (e.g., an oligonucleotide) can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the complementary strands, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. The nucleic acid alsocan be produced biologically using an expression vector into which anucleic acid has been subcloned.

One method uses at least two populations of oligonucleotide magneticnanoparticles, each with strong effects on water (or other solvent)relaxation. As the oligonucleotide-nanoparticle conjugates react with atarget oligonucleotide, they form aggregates (e.g. 100-500 nm). Uponprolonged standing, e.g., overnight at room temperature, the aggregatesform large clusters (micron-sized particles), which settle out ofsolution. Preferred embodiments use magnetic resonance to determine therelaxation properties of the solvent, which are altered when the mixtureof magnetic oligonucleotide nanoparticles reacts with a target nucleicacid to form aggregates.

Certain embodiments employ a mixture of at least two types of magneticmetal oxide nanoparticles, each with a specific sequence ofoligonucleotide, and each with more than one copy of the oligonucleotideattached, e.g., covalently, per nanoparticle. For example, the assayprotocol may involve preparing a mixture of populations ofoligonucleotide-nanoparticle conjugates and reacting the mixture with atarget nucleic acid. Alternatively, oligonucleotide-nanoparticleconjugates can be reacted with the target in a sequential fashion.Certain embodiments feature the use of magnetic resonance to detect thereaction of the oligonucleotide-nanoparticle conjugates with the targetnucleic acid. When a target is present, the dispersed conjugatesself-assemble to form small aggregates.

For example, oligonucleotide binding moieties can be linked to the metaloxide through covalent attachment to a functionalized polymer or tonon-polymeric surface-functionalized metal oxides. In the latter method,the nanoparticles can be synthesized according to the method of Albrechtet al., Biochimie, 80 (5-6): 379-90, 1998. Dimercapto-succinic acid iscoupled to the iron oxide and provides a carboxyl functional group.

In certain embodiments, oligonucleotides are attached to magneticnanoparticles via a functionalized polymer associated with the metaloxide. In some embodiments, the polymer is hydrophilic. In certainembodiments, the conjugates are made using oligonucleotides that haveterminal amino, sulfhydryl, or phosphate groups, and superparamagneticiron oxide nanoparticles bearing amino or carboxy groups on ahydrophilic polymer. There are several methods for synthesizing carboxyand amino derivatized-nanoparticles.

Polypeptide Binding Moieties

In certain embodiments, the binding moiety is a polypeptide (i.e., aprotein, polypeptide, or peptide), attached, using any of a variety ofchemistries, by a single covalent bond in such a manner so as to notaffect the biological activity of the polypeptide. In one embodiment,attachment is done through the thiol group of single reactive cysteineresidue so placed that its modification does not affect the biologicalactivity of the polypeptide. In this regard the use of linearpolypeptides, with cysteine at the C-terminal or N-terminal end,provides a single thiol in a manner similar to which alkanethiolsupplies a thiol group at the 3′ or 5′ end of an oligonucleotide.Similar bifunctional conjugation reagents, such as SPDP and reactingwith the amino group of the nanoparticle and thiol group of thepolypeptide, can be used with any thiol bearing binding moiety. Thetypes of polypeptides used as binding moieties can be antibodies,antibody fragments, and natural and synthetic polypeptide sequences, forexample. The peptide binding moieties generally have a binding partner,that is, a molecule to which they selectively bind.

Use of peptides as binding moieties offers several advantages. Forexample, the mass per binding site is low. For example, up to twenty 2kDa peptides can be attached to a nanoparticle, calculated assuming 2064iron atoms per nanoparticle. With larger binding moieties like proteins(generally greater than about 30 kDa) the same mass of attachedpolypeptide results in only approximately 1-4 binding moieties pernanoparticle. Also, polypeptides can be engineered to have uniquelyreactive residues, distal from the residues required for biologicalactivity, for attachment to the nanoparticle. The reactive residue canbe a cysteine thiol, an N-terminal amino group, a C-terminal carboxylgroup or a carboxyl group of aspartate or glutamate, etc. A singlereactive residue on the peptide is used to insure a unique site ofattachment. These design principles can be followed with chemicallysynthesized peptides or biologically produced polypeptides.

The binding moieties can also contain amino acid sequences fromnaturally occurring (wild-type) polypeptides or proteins. For example,the natural polypeptide may be a hormone, (e.g., a cytokine, a growthfactor), a serum protein, a viral protein (e.g., hemagglutinin), anextracellular matrix protein, a lectin, or an ectodomain of a cellsurface protein. In general, the resulting binding moiety-nanoparticleis used to measure the presence of analytes in a test media reactingwith the binding moiety.

Examples of protein hormones include: platelet-derived growth factor(PDGF) which binds the PDGF receptor; insulin-like growth factor-I and-II (Igf) which binds the Igf receptor; nerve growth factor (NOF) whichbinds the NGF receptor; fibroblast growth factor (FGF) which binds theFGF receptor (e.g., aFGF and bFGF); epidermal growth factor (EGF) whichbinds the EGF receptor; transforming growth factor (TGF, e.g.,TGF-.alpha. and TGF-.beta.) which bind the TGF receptor; erythropoietin,which binds the erythropoitin receptor; growth hormone (e.g., humangrowth hormone) which binds the growth hormone receptor; and proinsulin,insulin, A-chain insulin, and B-chain insulin, which all bind to theinsulin receptor.

Receptor binding moieties are useful for detecting and imaging receptorclustering on the surface of a cell. Useful ectodomains include those ofthe Notch protein, Delta protein, integrins, cadherins, and other celladhesion molecules.

Antibody Binding Moieties

Other polypeptide binding moieties include immunoglobulin bindingmoieties that include at least one immunoglobulin domain, and typicallyat least two such domains. An “immunoglobulin domain” refers to a domainof a antibody molecule, e.g., a variable or constant domain. An“immunoglobulin superfamily domain” refers to a domain that has athree-dimensional structure related to an immunoglobulin domain, but isfrom a non-immunoglobulin molecule. Immunoglobulin domains andimmunoglobulin superfamily domains typically include two .beta.-sheetsformed of about seven .beta.-strands, and a conserved disulphide bond(see, e.g., Williams and Barclay. 1988 Ann. Rev Immunol. 6:381-405).Proteins that include domains of the Ig superfamily domains include Tcell receptors, CD4, platelet derived growth factor receptor (PDGFR),and intercellular adhesion molecule (ICAM).

One type of immunoglobulin binding moiety is an antibody. The term“antibody,” as used herein, refers to a full-length, two-chainimmunoglobulin molecule and an antigen-binding portion and fragmentsthereof, including synthetic variants. A typical antibody includes twoheavy (H) chain variable regions (abbreviated herein as VH), and twolight (L) chain variable regions (abbreviated herein as VL). The VH andVL regions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (CDR), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDR's has been precisely defined(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917). Each VH and VL is composed of three CDR's and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An antibody can also include a constant region as part of a light orheavy chain. Light chains can include a kappa or lambda constant regiongene at the COOH-terminus (termed CL). Heavy chains can include, forexample, a gamma constant region (IgG1, IgG2, IgG3, IgG4; encoding about330 amino acids). A gamma constant region can include, e.g., CH1, CH2,and CH3. The term “full-length antibody” refers to a protein thatincludes one polypeptide that includes VL and CL, and a secondpolypeptide that includes VH, CH1, CH2, and CH3.

The term “antigen-binding fragment” of an antibody, as used herein,refers to one or more fragments of a full-length antibody that retainthe ability to specifically bind to a target. Examples ofantigen-binding fragments include, but are not limited to: (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsoencompassed within the term “antigen-binding fragment.”

In certain embodiments, the binding moiety is a polysaccharide, linked,for example, using any of a variety of chemistries, by a single bond,e.g., a covalent bond, at one of the two ends, to a functional group onthe nanoparticle. The polysaccharides can be synthetic or natural.Mono-, di-, tri- and polysaccharides can be used as the binding moiety.These include, e.g., glycosides, N-glycosylamines, O-acyl derivatives,O-methyl derivatives, osazones, sugar alcohols, sugar acids, sugarphosphates when used with appropriate attachment chemistry to thenanoparticle.

A method of accomplishing linking is to couple avidin to a magneticnanoparticle and react the avidin-nanoparticle with commerciallyavailable biotinylated polysaccharides, to yieldpolysaccharide-nanoparticle conjugates. For example, sialyl Lewis basedpolysaccharides are commercially available as biotinylated reagents andwill react with avidin-CLIO (see Syntesome, Gesellschaft furmedizinische Biochemie mbH.). The sialyl Lewis×tetrasaccharide (Sle^(X))is recognized by proteins known as selecting, which are present on thesurfaces of leukocytes and function as part of the inflammatory cascadefor the recruitment of leukocytes.

Still other targeting moieties include a non-proteinaceous element,e.g., a glycosyl modification (such as a Lewis antigen) or anothernon-proteinaceous organic molecule.

Biologically Active Substances

Embodiments of the invention include devices and/or systems fordetecting and/or measuring the concentration of one or more analytes ina sample. The analyte(s) may include one or more biologically activesubstances and/or metabolite(s), marker(s), and/or other indicator(s) ofbiologically active substances. A biologically active substance may bedescribed as a single entity or a combination of entities. The term“biologically active substance” includes without limitation,medications; vitamins; mineral supplements; substances used for thetreatment, prevention, diagnosis, cure or mitigation of disease orillness; or substances which affect the structure or function of thebody; or pro-drugs, which become biologically active or more activeafter they have been placed in a predetermined physiologicalenvironment.

Non-limiting examples of broad categories of useful biologically activesubstances include the following therapeutic categories: anabolicagents, antacids, anti -asthmatic agents, anti-cholesterolemic andanti-lipid agents, anti-coagulants, anti-convulsants, anti-diarrheals,anti-emetics, anti-infective agents, anti-inflammatory agents,anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesityagents, anti-pyretic and analgesic agents, anti-spasmodic agents,anti-thrombotic agents, anti-uricemic agents, anti-anginal agents,antihistamines, anti-tussives, appetite suppressants, biologicals,cerebral dilators, coronary dilators, decongestants, diuretics,diagnostic agents, erythropoietic agents, expectorants, gastrointestinalsedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ionexchange resins, laxatives, mineral supplements, mucolytic agents,neuromuscular drugs, peripheral vasodilators, psychotropics, sedatives,stimulants, thyroid and anti-thyroid agents, uterine relaxants,vitamins, and prodrugs.

More specifically, non-limiting examples of useful biologically activesubstances include the following therapeutic categories: analgesics,such as nonsteroidal anti-inflammatory drugs, opiate agonists andsalicylates; antihistamines, such as H₁-blockers and H₂-blockers;anti-infective agents, such as anthelmintics, antianaerobics,antibiotics, aminoglycoside antibiotics, antifungal antibiotics,cephalosporin antibiotics, macrolide antibiotics, miscellaneous.beta.-lactam antibiotics, penicillin antibiotics, quinoloneantibiotics, sulfonamide antibiotics, tetracycline antibiotics,antimycobacterials, antituberculosis antimycobacterials, antiprotozoals,antimalarial antiprotozoals, antiviral agents, antiretroviral agents,scabicides, and urinary anti-infectives; antineoplastic agents, such asalkylating agents, nitrogen mustard alkylating agents, nitrosoureaalkylating agents, antimetabolites, purine analog antimetabolites,pyrimidine analog antimetabolites, hormonal antineoplastics, naturalantineoplastics, antibiotic natural antineoplastics, and vinca alkaloidnatural antineoplastics; autonomic agents, such as anticholinergics,antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics,cholinergic agonist parasympathomimetics, cholinesterase inhibitorparasympathomimetics, sympatholytics, alpha-blocker sympatholytics,beta-blocker sympatholytics, sympathomimetics, and adrenergic agonistsympathomimetics; cardiovascular agents, such as antianginals,beta-blocker antianginals, calcium-channel blocker antianginals, nitrateantianginals, antiarrhythmics, cardiac glycoside antiarrhythmics, classI antiarrhythmics, class II antiarrhythmics, class III antiarrhythmics,class IV antiarrhythmics, antihypertensive agents, alpha-blockerantihypertensives, angiotensin-converting enzyme inhibitor (ACEinhibitor) antihypertensives, beta-blocker antihypertensives,calcium-channel blocker antihypertensives, central-acting adrenergicantihypertensives, diuretic antihypertensive agents, peripheralvasodilator antihypertensives, antilipemics, bile acid sequestrantantilipemics, HMG-COA reductase inhibitor antilipemics, inotropes,cardiac glycoside inotropes, and thrombolytic agents; dermatologicalagents, such as antihistamines, anti -inflammatory agents,corticosteroid anti-inflammatory agents, antipruritics/localanesthetics, topical anti-infectives, antifungal topicalanti-infectives, antiviral topical anti-infectives, and topicalantineoplastics; electrolytic and renal agents, such as acidifyingagents, alkalinizing agents, diuretics, carbonic anhydrase inhibitordiuretics, loop diuretics, osmotic diuretics, potassium-sparingdiuretics, thiazide diuretics, electrolyte replacements, and uricosuricagents; enzymes, such as pancreatic enzymes and thrombolytic enzymes;gastrointestinal agents, such as antidiarrheals, antiemetics,gastrointestinal anti-inflammatory agents, salicylate gastrointestinalanti-inflammatory agents, antacid anti-ulcer agents, gastric acid-pumpinhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents,H₂-blocker anti-ulcer agents, cholelitholytic agents, digestants,emetics, laxatives and stool softeners, and prokinetic agents; generalanesthetics, such as inhalation anesthetics, halogenated inhalationanesthetics, intravenous anesthetics, barbiturate intravenousanesthetics, benzodiazepine intravenous anesthetics, and opiate agonistintravenous anesthetics; hematological agents, such as antianemiaagents, hematopoietic antianemia agents, coagulation agents,anticoagulants, hemostatic coagulation agents, platelet inhibitorcoagulation agents, thrombolytic enzyme coagulation agents, and plasmavolume expanders; hormones and hormone modifiers, such asabortifacients, adrenal agents, corticosteroid adrenal agents,androgens, anti-androgens, antidiabetic agents, sulfonylureaantidiabetic agents, antihypoglycemic agents, oral contraceptives,progestin contraceptives, estrogens, fertility agents, oxytocics,parathyroid agents, pituitary hormones, progestins, antithyroid agents,thyroid hormones, and tocolytics; immunobiologic agents, such asimmunoglobulins, immunosuppressives, toxoids, and vaccines; localanesthetics, such as amide local anesthetics and ester localanesthetics; musculoskeletal agents, such as anti-gout anti-inflammatoryagents, corticosteroid anti-inflammatory agents, gold compoundanti-inflammatory agents, immuno-suppressive anti-inflammatory agents,nonsteroidal anti-inflammatory drugs (NSAIDs), salicylateanti-inflammatory agents, skeletal muscle relaxants, neuromuscularblocker skeletal muscle relaxants, and reverse neuromuscular blockerskeletal muscle relaxants; neurological agents, such as anticonvulsants,barbiturate anticonvulsants, benzodiazepine anticonvulsants,anti-migraine agents, anti-parkinsonian agents, anti-vertigo agents,opiate agonists, and opiate antagonists; ophthalmic agents, such asanti-glaucoma agents, beta-blocker anti-gluacoma agents, mioticanti-glaucoma agents, mydriatics, adrenergic agonist mydriatics,antimuscarinic mydriatics, ophthalmic anesthetics, ophthalmicanti-infectives, ophthalmic aminoglycoside anti-infectives, ophthalmicmacrolide anti-infectives, ophthalmic quinolone anti-infectives,ophthalmic sulfonamide anti-infectives, ophthalmic tetracyclineanti-infectives, ophthalmic anti-inflammatory agents, ophthalmiccorticosteroid anti-inflammatory agents, and ophthalmic nonsteroidalanti-inflammatory drugs (NSAIDs); psychotropic agents, such asantidepressants, heterocyclic antidepressants, monoamine oxidaseinhibitors (MAOIs), selective serotonin re-uptake inhibitors (SSRIs),tricyclic antidepressants, antimanics, antipsychotics, phenothiazineantipsychotics, anxiolytics, sedatives, and hypnotics, barbituratesedatives and hypnotics, benzodiazepine anxiolytics, sedatives, andhypnotics, and psychostimulants; respiratory agents, such asantitussives, bronchodilators, adrenergic agonist bronchodilators,antimuscarinic bronchodilators, expectorants, mucolytic agents,respiratory anti-inflammatory agents, and respiratory corticosteroidanti-inflammatory agents; toxicology agents, such as antidotes, heavymetal antagonists/chelating agents, substance abuse agents, deterrentsubstance abuse agents, and withdrawal substance abuse agents; minerals;and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D,vitamin E, and vitamin K.

Examples of classes of biologically active substances from the abovecategories include: nonsteroidal anti-inflammatory drugs (NSAIDs)analgesics, such as diclofenac, ibuprofen, ketoprofen, and naproxen;opiate agonist analgesics, such as codeine, fentanyl, hydromorphone, andmorphine; salicylate analgesics, such as aspirin (ASA) (enteric coatedASA); H₁-blocker antihistamines, such as clemastine and terfenadine;H₂-blocker antihistamines, such as cimetidine, famotidine, nizadine, andranitidine; anti-infective agents, such as mupirocin; antianaerobicanti-infectives, such as chloramphenicol and clindamycin; antifungalantibiotic anti-infectives, such as amphotericin b, clotrimazole,fluconazole, and ketoconazole; macrolide antibiotic anti -infectives,such as azithromycin and erythromycin; miscellaneous beta-lactamantibiotic anti-infectives, such as aztreonam and imipenem; penicillinantibiotic anti-infectives, such as nafcillin, oxacillin, penicillin G,and penicillin V; quinolone antibiotic anti -infectives, such asciprofloxacin and norfloxacin; tetracycline antibiotic anti-infectives,such as doxycycline, minocycline, and tetracycline; antituberculosisantimycobacterial anti-infectives such as isoniazid (INH), and rifampin;antiprotozoal anti-infectives, such as atovaquone and dapsone;antimalarial antiprotozoal anti-infectives, such as chloroquine andpyrimethamine; anti-retroviral anti-infectives, such as ritonavir andzidovudine; antiviral anti-infective agents, such as acyclovir,ganciclovir, interferon alfa, and rimantadine; alkylating antineoplasticagents, such as carboplatin and cisplatin; nitrosourea alkylatingantineoplastic agents, such as carmustine (BCNU); antimetaboliteantineoplastic agents, such as methotrexate; pyrimidine analogantimetabolite antineoplastic agents, such as fluorouracil (5-FU) andgemcitabine; hormonal antineoplastics, such as goserelin, leuprolide,and tamoxifen; natural antineoplastics, such as aldesleukin,interleukin-2, docetaxel, etoposide (VP-16), interferon alfa,paclitaxel, and tretinoin (ATRA); antibiotic natural antineoplastics,such as bleomycin, dactinomycin, daunorubicin, doxorubicin, andmitomycin; vinca alkaloid natural antineoplastics, such as vinblastineand vincristine; autonomic agents, such as nicotine; anticholinergicautonomic agents, such as benztropine and trihexyphenidyl;antimuscarinic anticholinergic autonomic agents, such as atropine andoxybutynin; ergot alkaloid autonomic agents, such as bromocriptine;cholinergic agonist parasympathomimetics, such as pilocarpine;cholinesterase inhibitor parasympathomimetics, such as pyridostigmine;alpha-blocker sympatholytics, such as prazosin; 9-blockersympatholytics, such as atenolol; adrenergic agonist sympathomimetics,such as albuterol and dobutamine; cardiovascular agents, such as aspirin(ASA) (enteric coated ASA); i-blocker antianginals, such as atenolol andpropranolol; calcium-channel blocker antianginals, such as nifedipineand verapamil; nitrate antianginals, such as isosorbide dinitrate(ISDN); cardiac glycoside antiarrhythmics, such as digoxin; class Iantiarrhythmics, such as lidocaine, mexiletine, phenyloin, procainamide,and quinidine; class II antiarrhythmics, such as atenolol, metoprolol,propranolol, and timolol; class III antiarrhythmics, such as amiodarone;class IV antiarrhythmics, such as diltiazem and verapamil; alpha-blockerantihypertensives, such as prazosin; angiotensin-converting enzymeinhibitor (ACE inhibitor) antihypertensives, such as captopril andenalapril; beta-blocker antihypertensives, such as atenolol, metoprolol,nadolol, and propanolol; calcium-channel blocker antihypertensiveagents, such as diltiazem and nifedipine; central-acting adrenergicantihypertensives, such as clonidine and methyldopa; diureticantihypertensive agents, such as amiloride, furosemide,hydrochlorothiazide (HCTZ), and spironolactone; peripheral vasodilatorantihypertensives, such as hydralazine and minoxidil; antilipemics, suchas gemfibrozil and probucol; bile acid sequestrant antilipemics, such ascholestyramine; HMG-CoA reductase inhibitor antilipemics, such aslovastatin and pravastatin; inotropes, such as amrinone, dobutamine, anddopamine; cardiac glycoside inotropes, such as digoxin; thrombolyticagents, such as alteplase (TPA), anistreplase, streptokinase, andurokinase; dermatological agents, such as colchicine, isotretinoin,methotrexate, minoxidil, tretinoin (ATRA); dermatological corticosteroidanti-inflammatory agents, such as betamethasone and dexamethasone;antifungal topical anti-infectives, such as amphotericin B,clotrimazole, miconazole, and nystatin; antiviral topicalanti-infectives, such as acyclovir; topical antineoplastics, such asfluorouracil (5-FU); electrolytic and renal agents, such as lactulose;loop diuretics, such as furosemide; potassium-sparing diuretics, such astriamterene; thiazide diuretics, such as hydrochlorothiazide (HCTZ);uricosuric agents, such as probenecid; enzymes such as RNase and DNase;thrombolytic enzymes, such as alteplase, anistreplase, streptokinase andurokinase; antiemetics, such as prochlorperazine; salicylategastrointestinal anti-inflammatory agents, such as sulfasalazine;gastric acid-pump inhibitor anti-ulcer agents, such as omeprazole;H₂-blocker anti-ulcer agents, such as cimetidine, famotidine,nizatidine, and ranitidine; digestants, such as pancrelipase; prokineticagents, such as erythromycin; opiate agonist intravenous anestheticssuch as fentanyl; hematopoietic antianemia agents, such aserythropoietin, filgrastim (G-CSF), and sargramostim (GM-CSF);coagulation agents, such as antihemophilic factors 1-10 (AHF 1-10);anticoagulants, such as warfarin; thrombolytic enzyme coagulationagents, such as alteplase, anistreplase, streptokinase and urokinase;hormones and hormone modifiers, such as bromocriptine; abortifacients,such as methotrexate; antidiabetic agents, such as insulin; oralcontraceptives, such as estrogen and progestin; progestincontraceptives, such as levonorgestrel and norgestrel; estrogens such asconjugated estrogens, diethylstilbestrol (DES), estrogen (estradiol,estrone, and estropipate); fertility agents, such as clomiphene, humanchorionic gonadatropin (HCG), and menotropins; parathyroid agents suchas calcitonin; pituitary hormones, such as desmopressin, goserelin,oxytocin, and vasopressin (ADH); progestins, such asmedroxyprogesterone, norethindrone, and progesterone; thyroid hormones,such as levothyroxine; immunobiologic agents, such as interferon beta-Iband interferon gamma-Ib; immunoglobulins, such as immune globulin IM,IMIG, IGIM and immune globulin IV, IVIG, IGIV; amide local anesthetics,such as lidocaine; ester local anesthetics, such as benzocaine andprocaine; musculoskeletal corticosteroid anti-inflammatory agents, suchas beclomethasone, betamethasone, cortisone, dexamethasone,hydrocortisone, and prednisone; musculoskeletal anti-inflammatoryimmunosuppressives, such as azathioprine, cyclophosphamide, andmethotrexate; musculoskeletal nonsteroidal anti-inflammatory drugs(NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketorlac, andnaproxen; skeletal muscle relaxants, such as baclofen, cyclobenzaprine,and diazepam; reverse neuromuscular blocker skeletal muscle relaxants,such as pyridostigmine; neurological agents, such as nimodipine,riluzole, tacrine and ticlopidine; anticonvulsants, such ascarbamazepine, gabapentin, lamotrigine, phenyloin, and valproic acid;barbiturate anticonvulsants, such as phenobarbital and primidone;benzodiazepine anticonvulsants, such as clonazepam, diazepam, andlorazepam; anti-parkisonian agents, such as bromocriptine, levodopa,carbidopa, and pergolide; anti-vertigo agents, such as meclizine; opiateagonists, such as codeine, fentanyl, hydromorphone, methadone, andmorphine; opiate antagonists, such as naloxone; beta-blockeranti-glaucoma agents, such as timolol; miotic anti-glaucoma agents, suchas pilocarpine; ophthalmic aminoglycoside antiinfectives, such asgentamicin, neomycin, and tobramycin; ophthalmic quinoloneanti-infectives, such as ciprofloxacin, norfloxacin, and ofloxacin;ophthalmic corticosteroid anti-inflammatory agents, such asdexamethasone and prednisolone; ophthalmic nonsteroidalanti-inflammatory drugs (NSAIDs), such as diclofenac; antipsychotics,such as clozapine, haloperidol, and risperidone; benzodiazepineanxiolytics, sedatives and hypnotics, such as clonazepam, diazepam,lorazepam, oxazepam, and prazepam; psychostimulants, such asmethylphenidate and pemoline; antitussives, such as codeine;bronchodilators, such as theophylline; adrenergic agonistbronchodilators, such as albuterol; respiratory corticosteroidanti-inflammatory agents, such as dexamethasone; antidotes, such asflumazenil and naloxone; heavy metal antagonists/chelating agents, suchas penicillamine; deterrent substance abuse agents, such as disulfiram,naltrexone, and nicotine; withdrawal substance abuse agents, such asbromocriptine; minerals, such as iron, calcium, and magnesium; vitamin Bcompounds, such as cyanocobalamin (vitamin B₁₂) and niacin (vitamin B₃);vitamin C compounds, such as ascorbic acid; and vitamin D compounds,such as calcitriol; recombinant beta-glucan; bovine immunoglobulinconcentrate; bovine superoxide dismutase; the formulation comprisingfluorouracil, epinephrine, and bovine collagen; recombinant hirudin(r-Hir), HIV-1 immunogen; human anti-TAC antibody; recombinant humangrowth hormone (r-hGH); recombinant human hemoglobin (r-Hb); recombinanthuman mecasermin (r-IGF-1); recombinant interferon beta-1a; lenograstim(G-CSF); olanzapine; recombinant thyroid stimulating hormone (r-TSH);topotecan; acyclovir sodium; aldesleukin; atenolol; bleomycin sulfate,human calcitonin; salmon calcitonin; carboplatin; carmustine;dactinomycin, daunorubicin HCl; docetaxel; doxorubicin HCl; epoetinalfa; etoposide (VP-16); fluorouracil (5-FU); ganciclovir sodium;gentamicin sulfate; interferon alfa; leuprolide acetate; meperidine HCl;methadone HCl; methotrexate sodium; paclitaxel; ranitidine HCl;vinblastin sulfate; and zidovudine (AZT).

Further specific examples of biologically active substances from theabove categories include: antineoplastics such as androgen inhibitors,antimetabolites, cytotoxic agents, and immunomodulators; anti-tussivessuch as dextromethorphan, dextromethorphan hydrobromide, noscapine,carbetapentane citrate, and chlorphedianol hydrochloride; antihistaminessuch as chlorpheniramine maleate, phenindamine tartrate, pyrilaminemaleate, doxylamine succinate, and phenyltoloxamine citrate;decongestants such as phenylephrine hydrochloride, phenylpropanolaminehydrochloride, pseudoephedrine hydrochloride, and ephedrine; variousalkaloids such as codeine phosphate, codeine sulfate and morphine;mineral supplements such as potassium chloride, zinc chloride, calciumcarbonates, magnesium oxide, and other alkali metal and alkaline earthmetal salts; ion exchange resins such as cholestryramine;anti-arrhythmics such as N-acetylprocainamide; antipyretics andanalgesics such as acetaminophen, aspirin and ibuprofen; appetitesuppressants such as phenyl-propanolamine hydrochloride or caffeine;expectorants such as guaifenesin; antacids such as aluminum hydroxideand magnesium hydroxide; biologicals such as peptides, polypeptides,proteins and amino acids, hormones, interferons or cytokines, and otherbioactive peptidic compounds, such as interleukins 1-18 includingmutants and analogues, RNase, DNase, luteinizing hormone releasinghormone (LHRH) and analogues, gonadotropin releasing hormone (GnRH),transforming growth factor-beta(TGF-beta), fibroblast growth factor(FGF), tumor necrosis factor-alpha & beta (TNF-alpha & beta), nervegrowth factor (NGF), growth hormone releasing factor (GHRF), epidermalgrowth factor (EGF), fibroblast growth factor homologous factor (FGFHF),hepatocyte growth factor (HGF), insulin growth factor (IGF), invasioninhibiting factor-2 (IIF-2), bone morphogenetic proteins 1-7 (BMP 1-7),somatostatin, thymosin-.alpha.-1, T-globulin, superoxide dismutase(SOD), complement factors, hGH, tPA, calcitonin, ANF, EPO and insulin;and anti-infective agents such as antifungals, anti-virals, antisepticsand antibiotics.

Biologically active substances also include radiosensitizers, such asmetoclopramide, sensamide or neusensamide (manufactured by Oxigene);profiromycin (made by Vion); RSR13 (made by Allos); Thymitaq (made byAgouron), etanidazole or lobenguane (manufactured by Nycomed);gadolinium texaphrin (made by Pharmacyclics); BuDR/Broxine (made byNeoPharm); IPdR (made by Sparta); CR2412 (made by Cell Therapeutic); LIX(made by Terrapin); or the like.

Biologically active substances include medications for thegastrointestinal tract or digestive system, for example, antacids,reflux suppressants, antiflatulents, antidoopaminergics, proton pumpinhibitors, H2-receptor antagonists, cytoprotectants, prostaglandinanalogues, laxatives, antispasmodics, antidiarrheals, bile acidsequestrants, and opioids; medications for the cardiovascular system,for example, beta-receptor blockers, calcium channel blockers,diuretics, cardiac glycosides, antiarrhythmics, nitrate, antianginals,vascoconstrictors, vasodilators, peripheral activators, ACE inhibitors,angiotensin receptor blockers, alpha blockers, anticoagulants, heparin,HSGAGs, antiplatelet drugs, fibrinolytics, anti-hemophilic factors,haemostatic drugs, hypolipaemic agents, and statins; medications for thecentral nervous system, for example, hypnotics, anaesthetics,antipsychotics, antidepressants, anti-emetics, anticonvulsants,antiepileptics, anxiolytics, barbiturates, movement disorder drugs,stimulants, benzodiazepine, cyclopyrrolone, dopamine antagonists,antihistamine, cholinergics, anticholinergics, emetics, cannabinoids,5-HT antigonists; medications for pain and/or consciousness, forexample, NSAIDs, opioids and orphans such as paracetamol, tricyclicantidepressants, and anticonvulsants; for musculo-skeletal disorders,for example, NSAIDs, muscle relaxants, and neuromuscular druganticholinersterase; medications for the eye, for example, adrenergicneurone blockers, astringents, ocular lubricants, topical anesthetics,sympathomimetics, parasympatholytics, mydriatics, cycloplegics,antibiotics, topical antibiotics, sulfa drugs, aminoglycosides,fluoroquinolones, anti-virals, anti-fungals, imidazoles, polyenes,NSAIDs, corticosteroids, mast cell inhibitors, adrenergic agonists,beta-blockers, carbonic anhydrase inhibitors/hyperosmotiics,cholinergics, miotics, parasympathomimetics, prostaglandin,agonists/prostaglandin inhibitors, nitroglycerin; medications for theear, nose and oropharynx, for example, sympathomimetics, antihistamines,anticholinergics, NSAIDs, steroids, antiseptics, local anesthetics,antifungals, cerumenolytics; medications for the respiratory system, forexample, bronchodilators, NSAIDs, anti-allergics, antitussives,mucolytics, decongestants, corticosteroids, beta-receptor antagonists,anticholinergics, steroids; medications for endocrine problems, forexample, androgen, antiandrogen, gonadotropin, corticosteroids, growthhormone, insulin, antidiabetics, thyroid hormones, antithyroid drugs,calcitonin, diphosphonate, and vasopressin analogues; medications forthe reproductive system or urinary system, for example, antifungals,alkalising agents, quinolones, antibiotics, cholinergics,anticholinergics, anticholinesterase, antispasmodics, 5-alpha reductaseinhibitor, selective alpha-1 blockers, and sildenafil; medications forcontraception, for example, oral contraceptives, spermicides, and depotcontraceptives; medications for obstetrics and gynecology, for example,NSAIDs, anticholinergics, haemostatic drugs, antifibrinolytics, hormonereplacement therapy, bone regulator, beta-receptor agonists, folliclestimulating hormone, luteinising hormone, LHRH gamolenic acid,gonadotropin release inhibitor, progestogen, dopamine agonist,oestrogen, prostaglandin, gonadorelin, clomiphene, tammoxifen, anddiethylstilbestrol; medications for the skin, for example, emollients,anti-pruritics, antifungals, disinfectants, scabicide, pediculicide, tarproducts, vitamin A derivatives, vitamin D analogue, keratolytics,abrasives, systemic antibiotics, topical antibiotics, hormones,desloughing agents, exudate absorbents, fibrinolytics, proteolytics,sunscreens, antiperspirants, and corticosteroids; medications forinfections and infestations, for example, antibiotics, antifungals,antileprotics, antituberculous drugs, antimalarials, anthelmintics,amoebicide, antivirals, antiprotozoals, and antiserum; medications forthe immune system, for example, vaccines, immunoglobulin,immunosuppressants, interferon, monoclonal antibodies; medications forallergic disorders, for example, anti-allergies, antihistamines, andNSAIDs; medications for nutrition, for example, tonics, ironpreparations, electrolytes, vitamins, anti-obesity drugs, anabolicdrugs, haematopoietic drugs, and food product drugs; medications forneoplastic disorders, for example, cytotoxic drugs, sex hormones,aromatase inhibitors, somatostatin inhibitors, recombinant interleukins,G-CSF, and erythropoietin; medications for diagnostics, for example,contrast agents; and medications for cancer (anti-cancer agents).

Examples of pain medications (e.g. analgesics) include opioids such asbuprenorphine, butorphanol, dextropropoxyphene, dihydrocodeine,fentanyl, diamorphine (heroin), hydromorphone, morphine, nalbuphine,oxycodone, oxymorphone, pentazocine, pethidine (meperidine), andtramadol; salicylic acid and derivatives such as acetylsalicylic acid(aspirin), diflunisal, and ethenzamide; pyrazolones such asaminophenazone, metamizole, and phenazone; anilides such as paracetamol(acetaminophen), phenacetin; and others such as ziconotide andtetradyrocannabinol.

Examples of blood pressure medications (e.g. antihypertensives anddiuretics) include antiadrenergic agents such as clonidine, doxazosin,guanethidine, guanfacine, mecamylamine, methyldopa, moxonidinie,prazosin, rescinnamine, and reserpine; vasodilators such as diazoxide,hydralazine, minoxidil, and nitroprusside; low ceiling diuretics such asbendroflumethiazide, chlorothiazide, chlortalidone, hydrochlorothiazide,indapamide, quinethazone, mersalyl, metolazone, and theobromine; highceiling diuretics such as bumetanide, furosemide, and torasemide;potassium-sparing diuretics such as amiloride, eplerenone,spironolactone, and triamterene; and other antihypertensives such asbosentan and ketanserin.

Examples of anti-thrombotics (e.g. thrombolytics, anticoagulants, andantiplatelet drugs) include vitamin K antagonists such as acenocoumarol,clorindione, dicumarol, diphenadione, ethyl biscoumacetate,phenprocoumon, phenindione, tioclomarol, and warfarin; heparin group(platelet aggregation inhibitors) such as antithrombin III, bemiparin,dalteparin, danaparoid, enoxaparin, heparin, nadroparin, parnaparin,reviparin, sulodexide, and tinzaparin; other platelet aggregationinhibitors such as abciximab, acetylsalicylic acid (aspirin), aloxiprin,beraprost, ditazole, carbasalate calcium, cloricromen, clopidogrel,dipyridamole, epoprostenol, eptifibatide, indobufen, iloprost,picotamide, prasugrel, ticlopidine, tirofiban, treprostinil, andtriflusal; enzymes such as alteplase, ancrod, anistreplase, brinase,drotrecogin alfa, fibrinolysin, procein C, reteplase, saruplase,streptokinase, tenecteplase, and urokinase; direct thrombin inhibitorssuch as argatroban, bivalirudin, desirudin, lepirudin, melagatran, andximelagatran; other antithrombotics such as dabigatran, defibrotide,dermatan sulfate, fondaparinux, and rivaroxaban; and others such ascitrate, EDTA, and oxalate.

Examples of anticonvulsants include barbiturates such as barbexaclone,metharbital, methylphenobarbital, phenobarbital, and primidone;hydantoins such as ethotoin, fosphenytoin, mephenyloin, and phenyloin;oxazolidinediones such as ethadione, paramethadione, and trimethadione;succinimides such as ethosuximide, mesuximide, and phensuximide;benzodiazepines such as clobazam, clonazepam, clorazepate, diazepam,lorazepam, midazolam, and nitrazepam; carboxamides such ascarbamazepine, oxcarbazepine, rufinamide; fatty acid derivatives such asvalpromide and valnoctamide; carboxylic acids such as valproic acid,tiagabine; GABA analogs such as gabapentin, pregabalin, progabide, andgivabatrin; monosaccharides such as topiramate; aromatic allyllicalcohols such as stiripentol; ureas such as phenacemide and pheneturide;carbamates such as emylcamate, felbamate, and meprobamate; pyrrolidinessuch as brivaracetam, levetiracetam, nefiracetam, and seletracetam;sulfa drugs such as acetazolamide, ethoxzolamide, sultiame, andzonisamide; propionates such as beclamide; aldehydes such asparaldehyde; and bromides such as potassium bromide.

Examples of anti-cancer agents include acivicin; aclarubicin; acodazolehydrochloride; acronine; adriamycin; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecanhydrochloride; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; Uracil mustard; rredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other biologically active substances include those mentioned in Basicand Clinical Pharmacology (LANGE Basic Science), Katzung and Katzung,ISBN 0071410929, McGraw-Hill Medical, 9^(th) edition (2003).

Medical Conditions

Embodiments of the invention may be used in the monitoring of one ormore biologically active substance(s) in the diagnosis, management,and/or treatment of any of a wide range of medical conditions. Variouscategories of medical conditions include, for example, disorders ofpain; of alterations in body temperature (e.g., fever); of nervoussystem dysfunction (e.g., syncope, myalgias, movement disorders,numbness, sensory loss, delirium, dimentioa, memory loss, sleepdisorders); of the eyes, ears, nose, and throat; of circulatory and/orrespiratory functions (e.g., dysplnea, pulmonary edema, cough,hemoptysis, hypertension, myocardial infarctions, hypoxia, cyanosis,cardiovascular collapse, congestive heart failure, edema, shock); ofgastrointestinal function (e.g., dysphagia, diarrhea, constipation, GIbleeding, jaundice, ascites, indigestion, nausea, vomiting); of renaland urinary tract function (e.g., acidosis, alkalosis, fluid andelectrolyte imbalances, azotemia, urinary abnormalities); of sexualfunction and reproduction (e.g., erectile dysfunction, menstrualdisturbances, hirsutism, virilization, infertility, pregnancy associateddisorders and standard measurements); of the skin (e.g., eczema,psoriasis, acne, rosacea, cutaneous infection, immunological skindiseases, photosensitivity); of the blood (e.g., hematology); of genes(e.g., genetic disorders); of drug response (e.g., adverse drugresponses); and of nutrition (e.g., obesity, eating disorders,nutritional assessment). Other medical fields with which embodiments ofthe invention find utility include oncology (e.g., neoplasms,malignancies, angiogenesis, paraneoplasic syndromes, oncologicemergencies); hematology (e.g., anemia, hemoglobinopathies,megalooblastic anemias, hemolytic anemias, aplastic anemia,myelodysplasia, bone marrow failure, polycythemia vera,myloproliferative diseases, acute myeloid leukemia, chronic myeloidleukemia, lymphoid malignancies, plasma cell disorders, transfusionbiology, transplants); hemostasis (e.g., disorders of coagulation andthrombosis, disorders of the platelet and vessel wall); and infectiousdiseases (e.g., sepsis, septic shock, fever of unknown origin,endocarditis, bites, burns, osteomyelitis, abscesses, food poisoning,peliv inflammatory disease, bacterial (gram positive, gram negative,miscellaneous (nocardia, actimoyces, mixed), mycobacterial, spirochetal,rickettsia, mycoplasma); chlamydia; viral (DNA, RNA), fungal and algalinfections; protozoal and helminthic infections; endocrine diseases;nutritional diseases; and metabolic diseases.

Other medical conditions and/or fields with which embodiments of theinvention find utility include those mentioned in Harrison's Principlesof Internal Medicine, Kasper et al., ISBN 0071402357, McGraw-HillProfessional, 16^(th) edition (2004), as well as those mentioned inRobbins Basic Pathology, Kumar, Cotran, and Robbins, eds., ISBN1416025340, Elsevier, 7^(th) edition (2005).

Medical tests (e.g. blood tests, urine tests, and/or other human oranimal tissue tests) that may be performed using various embodiments ofthe invention described herein include, for example, general chemistrytests (e.g., analytes include albumin, blood urea nitrogen, calcium,creatinine, magnesium, phosphorus, total protein, and/or uric acid);electrolyte tests (e.g., analytes include sodium, potassium, chloride,and/or carbon dioxide); diabetes tests (e.g., analytes include glucose,hemoglobin A1C, and/or microalbumin); lipids tests (e.g., analytesinclude apolipoprotein A1, apolipoprotein B, cholesterol, triglyceride,low density lipoprotein cholesterol, and/or high density lipoproteincholesterol); nutritional assessment (e.g., analytes include albumin,prealbumin, transferrin, retinol binding protein, alpha1-acidglycoprotein, and/or ferritin); hepatic tests (e.g., analytes includealanine transaminase, albumin, alkaline phosphatase, aspartatetransaminase, direct bilirubin, gamma glutamyl transaminase, lactatedehydrogenase, immunoglobulin A, immunoglobulin G, immunoglobulin M,prealbumin, total bilirubin, and/or total protein); cardiac tests (e.g.,analytes include apolipoprotein A1, apolipoprotein B, cardiactroponin-1, creatine kinase, creatine kinase MB isoenzyme, highsensitivity CRP, mass creatine kinase MB isoenzyme myoglobin, and/orN-terminal pro-brain natriuretic peptide); tests for anemia (e.g.,analytes include ferritin, folate, homocysteine, haptoglobin, iron,soluble transferrin receptor, total iron binding capacity, transferrin,and/or vitamin B12); pancreatic tests (e.g., analytes include amylaseand/or lipase); nephropathies (e.g., analytes include albumin,alpha1-microglobulin, alpha2-macroglobulin, beta2-microglobulin,cystatin C, retinol binding protein, and/or transferrin); bone tests(e.g., analytes include alkaline phosphatase, calcium, and/orphosphorous); cancer marker monitoring (e.g., analytes include totalPSA); thyroid tests (e.g., analytes include free thyroxine, freetriiodothyronine, thyroxine, thyroid stimulating hormone, and/ortriiodothyronine); fertility tests (e.g., analytes include beta-humanchorionic gonadotropin); therapeutic drug monitoring (e.g., analytesinclude carbamazepine, digoxin, digitoxin, gentamicin, lidocaine,lithium, N-acetyl procainamide, phenobarbital, phenyloin, procainamide,theophylline, tobramycin, valproic acid, and/or vancomycin);immunosuppressive drugs (e.g., analytes include cyclosporine A,sirolimus, and/or tacrolimus); tests for complement activity and/orautoimmune disease (e.g., analytes include C3 complement, C4 complement,C1 inhibitor, C-reactive protein, and/or rheumatoid factor);polyclonal/monoclonal gammopathies (e.g., analytes includeimmunoglobulin A, immunoglobulin G, immunoglobulin M, 1 g light chainstypes kappa and/or lambda, immunoglobulin G subclasses 1, 2, 3, and/or4); tests for infectious disease (e.g., analytes includeantistreptolysin 0); tests for inflammatory disorders (e.g., analytesinclude alpha1-acid glycoprotein, alpha1-antitrypsin, ceruloplasmin,C-reactive protein, and/or haptoglobin); allergy testing (e.g., analytesinclude immunoglobulin E); urine protein tests (e.g., analytes includealpha1-microglobulin, immunoglobulin G, 1 g light chans type kappaand/or lambda, microalbumin, and/or urinary/cerebrospinal fluidprotein); tests for protein—CSF (e.g., analytes include immunoglobulin Gand/or urinary/cerebrospinal fluid protein); toxicology tests (e.g.,analytes include serum acetaminophen, serum barbiturates, serumbenzodiazepines, serum salicylate, serum tricyclic antidepressants,and/or urine ethyl alcohol); and/or tests for drugs of abuse (e.g.,analytes include amphetamine, cocaine, barbiturates, benzodiazepines,ecstacy, methadone, opiate, phencyclidine, tetrahydrocannabinoids,propoxyphene, and/or methaqualone). In certain embodiments, the NMRdevice may replace large, expensive integrated analyzers, for example,those that integrate chemiluminescence, nephelometry, photometry, and/ormultisensor technologies. Other analytes include those mentioned in theTietz Textbook of Clinical Chemistry and Molecular Diagnostics, Burtis,Ashwood, and Bruns, ISBN 0721601898, Elsevier, 4th edition (2006).

NMR Systems/Devices

FIG. 1 is a schematic diagram 100 of an NMR system for detection of anecho response of a liquid sample to an RF excitation, thereby detectingthe presence and/or concentration of an analyte in the liquid sample. Abias magnet 102 establishes a bias magnetic field Bb 104 through asample 106. The nanoparticles are in a lyophilized state in the samplewell (the term “well” as used herein includes any indentation,container, or support) 108 until introduction of the liquid sample 106into the well 108, or the nanoparticles can be added to the sample 106prior to introduction of the liquid sample into the well 108. An RF coil110 and RF oscillator 112 provides an RF excitation at the Larmorfrequency which is a linear function of the bias magnetic field Bb. TheRF coil 110 is wrapped around the sample well 108. The excitation RFcreates instability in the spin of the water protons (or free protons ina non-aqueous solvent). When the RF excitation is turned off, theprotons “relax” to their original state and emit an RF signalcharacteristic of the concentration of the analyte. The coil 110 acts asan RF antenna and detects an “echo” of the relaxation. The echo ofinterest is the decay in time (generally 10-300 milliseconds) and iscalled the T2 signal. The RF signal from the coil 110 is amplified 114and processed to determine the T2 (decay time) response to theexcitation in the bias field Bb. The well 108 may be a small capillarytube with microliters of the analyte and a microcoil wound around it.Alternatively, the coil may be configured as shown in any of FIGS. 2A-Eabout or in proximity to the well.

FIGS. 2A-E illustrate micro NMR coil (RF coil) designs. FIG. 2A shows awound solenoid micro coil 200 about 100 μm in length. FIG. 2B shows a“planar” coil 202 (the coil is not truly planar, since the coil hasfinite thickness) about 1000 μm in diameter. FIG. 2C shows a MEMSsolenoid coil 204 about 100 μm×500 μm length×width and defining a volumeof about 0.02 μL. FIG. 2D shows a schematic of a MEMS Helmholz coil 206configuration, and FIG. 2E shows a schematic of a saddle coil 220configuration.

A wound solenoid micro coil 200 used for traditional NMR (non-MRS)detection is described in Seeber et al., “Design and testing of highsensitivity micro -receiver coil apparatus for nuclear magneticresonance and imaging,” Ohio State University, Columbus, Ohio. A planarmicro coil 202 used for traditional NMR detection is described in Massinet al., “High Q factor RF planar microcoil for micro-scale NMRspectroscopy,” Sensors and Actuators A 97-98, 280-288 (2002). AHelmholtz coil configuration 206 features a well 208 for holding asample, a top Si layer 210, a bottom Si layer 212, and deposited metalcoils 214. An example of a Helmholtz coil configuration 206 used fortraditional NMR detection is described in Syms et al, “MEMS HelmholzCoils for Magnetic Resonance Spectroscopy,” Journal of Micromechanicsand Micromachining 15 (2005) S1-S9.

The coil configuration may be chosen or adapted for specificimplementation of the micro-NMR-MRS technology, since different coilconfigurations offer different performance characteristics. For example,each of these coil geometries has a different performance and fieldalignment. The planar coil 202 has an RF field perpendicular to theplane of the coil. The solenoid coil 200 has an RF field down the axisof the coil, and the Helmholtz coil 206 has an RF field transverse tothe two rectangular coils 214. The Helmholtz 206 and saddle coils 220have transverse fields which would allow the placement of the permanentmagnet bias field above and below the well. Helmholtz 206 and saddlecoils 220 may be most effective for the chip design, while the solenoidcoil 200 may be most effective when the sample and MRS nanoparticles areheld in a micro tube.

The micro-NMR devices may be fabricated by winding or printing the coilsor by microelectromechanical system (MEMS) semiconductor fabricationtechniques. For example, a wound or printed coil/sample well module maybe about 100 μm in diameter, or as large as a centimeter or more. A MEMSunit or chip (thusly named since it is fabricated in a semiconductorprocess as a die on a wafer) may have a coil that is from about 10 μm toabout 1000 μm in characteristic dimension, for example. The wound orprinted coil/sample well configuration is referenced herein as a moduleand the MEMS version is referenced herein as a chip. For example, theliquid sample 108 may be held in a tube (for example, a capillary,pipette, or micro tube) with the coil wound around it, or it may be heldin wells on the chip with the RF coil surrounding the well.

FIG. 3 is a schematic diagram 300 of an NMR system employing magneticnanoparticles in a micro well 302 for holding a liquid sample, the well302 surrounded by an RF coil 304 on a substrate (chip, support) 306,where a magnet 308 for creating the bias magnetic field lies on thesubstrate 306. The micro NMR unit 300 may be manufactured using MEMStechnology. The well 302 containing the MRS nanoparticles is surroundedby an RF coil 304 which is in turn surrounded by the bias field magnet308. The permanent magnet sits on a substrate 306. The electronics 310for the amplification and/or other conditioning of the signal are shownin close proximity to the RF coil 304. This configuration may befabricated in a MEMS silicon process wherein the coil 304 and magnet 308are deposited on the surface of the chip and the electronics 310 aremade using standard semiconductor manufacturing techniques.

FIG. 4A is a schematic diagram 400 of an NMR system employing magneticnanoparticles in a micro well 402, where the magnet 404 for creating atop-to-bottom bias magnetic field does not lie on the chip. The magnet404 is above and below the well 402. The bias field 406 is created byexternal magnets 404. In order to achieve the high bias magnetic field406 required for NMR, the bias magnets 404 should be in very closeproximity to the well 402 and RF coil 408. This can be accomplished withthe micro NMR design, since the dimensions are very small and thepermanent magnet can be brought to within 1 mm or less of the well/coil.In this configuration the RF coil may be chosen as a Helmholtz 206 orsaddle coil 220 with its primary RF field 410 perpendicular to the biasfield 406 created by the two magnets 404. In this configuration the RFcoil 408 on the chip provides both the RF excitation and the RF echosense. The circuitry 412 must switch between excitation mode and sensemode.

FIG. 4B is a schematic diagram of an NMR system 420 employing magneticnanoparticles in a micro well 402, where the magnet 404 for creating aside-to-side bias magnetic field does not lie on the chip. The magnet404 is adjacent to the well 402.

FIG. 5A is a schematic diagram of an NMR system 500 including a singlewell 402 with external RF excitation coil 502. The magnet 404 may beexternal to the chip, or the magnet 404 may be attached to the chip. TheRF excitation in this configuration is provided by the separate andexternal RF coil 502. This allows for optimization of the excitation RFcoil 502 separate from the sense coil 408 on the chip which may beconstrained by fabrication limitations (e.g., choice of material,thickness, cross-section, and the like). In this configuration theexcitation field is produced by a solenoid 502 winding outside of themicro-NMR unit, which creates a field perpendicular to the bias fieldcreated by the bias magnet 404 and in the plane of the RF sense coil408.

A module approach (as contrasted to the MEMS approach) presents aminiaturization of the NMR configuration 100 of FIG. 1 . A liquid samplewith the MRS nanoparticles is held in a small tube 108 with the solenoidRF coil 110 wrapped around it and placed within the bias field 104. Theadvantage of this system with respect to a MEMS system is that largerquantities of sample, or physically larger analyte(s) and/or MRSparticles.

A panel or array of numerous well/coil units may be used in variousembodiments. The panel can have duplicate assay/nanoparticles to enhancesensitivity, accuracy, and/or repeatability of the analyte detectionand/or analyte concentration measurements. Multiple assays can perform avariety of diagnostic tests simultaneously. FIG. 5B is a schematicdiagram 520 of an NMR system including an array of wells 522 withexternal RF excitation coil 502 and external bias magnet 404.

Multi-well configurations are shown in FIGS. 6A-D. FIG. 6A shows asingle well/coil pair 600. The single well/coil pair 600 is repeated asmany times as desired, as shown in the multiple well array 610 in FIG.6B. FIG. 6C is a schematic 611 of an NMR system including multiple wellscontaining different nanoparticles customized for detection of differentanalytes. Different assay nanoparticles are placed in each well 612,614, 616, 618 to create a test of different analytes. FIG. 6D is aschematic 620 of an NMR system including groups of wells with identicalnanoparticles for obtaining multiple data points (redundantmeasurements) for increased precision, sensitivity, and/orrepeatability. Certain assays are duplicated—for example, wells 622,624, 626, 628 for detection of analyte A, and wells 630, 632, 634 fordetection of analyte B, for increased precision, sensitivity, and/orrepeatability necessary for certain tests.

FIG. 7 is a block diagram depicting basic components of an NMR system700, including electrical components. The sensor (relaxometer T2 sensor)702 provides the relaxation echo from the sample well 704 to the signalprocessing unit 706 while the excitation RF is provided by the RFgenerator 708.

FIG. 8 is a block diagram of an NMR system 800 including multiple wellsand sensing coils and an external RF excitation coil. The block diagramincludes the basic circuit elements in this configuration. The RFsensing coils and associated passives are represented at 810, where theassociated passives include inductors, resistors and/or capacitors forthe appropriate frequency response from the corresponding well. Eachsignal is amplified by an on-chip amplifier 820 and either ismultiplexed 830 to the off-chip processor 840 or is sequentiallyswitched 860 to the off-chip processor 840. The switching is practicalbecause, for example, with 100 sample wells in sequence, the elapsedprocessing time would be about 50 seconds or less with a single echopulse lasting about 500 ms. The off chip processor 840 manages the dataand performs both time domain 842 and frequency domain 844 analysis todetect the effects of the nanoparticle aggregation. An RF generator 850drives the external RF coil 860 at the appropriate Larmor frequency toproduce the bias magnet field. The RF generator 850 may or may not becontrolled by the off chip processor 840.

FIG. 9 is a block diagram of an NMR system 900 including multiple wellsand sensing coils, but without an external RF excitation coil (thesensing coils also serve as excitation coils). The on-chip elements arethe same as in FIG. 8 except that the RF excitation signal must passthrough the switch 830 and by pass 815 the amplifier 820 and associatedcircuitry to go directly to the coil 810.

The off chip processing may be performed in a reader or similar handheldor desktop device containing the time and frequency domain analysis andthe RF generator. The reader may also contain the bias field permanentmagnets and/or the RF excitation coil. FIG. 10 is a schematic diagram ofthe chip or module receiver/reader 1000. The chip or module ispositioned onto a sample plate 1002, which is inserted into the slotbetween the bias field permanent magnets 1004 and within the externalexcitation coil (if used) 1006. A mechanical slide 1008 is used to pushthe assay chip or module 1002 into the test slot between the permanentmagnets 1004. The reader 1000 may be partially or entirely housed in acase 1010, and may feature an input keypad 1012 and/or a display 1014.

FIG. 11 is a schematic diagram of a magnetic analyte concentrator 1100,which takes advantage of the effect of the MRS nanoparticles todifferentially move target aggregations in the intense magnetic field.The bias magnetic field 1102 will preferentially move the targetmolecules trapped in the aggregation of the magnetic nanoparticles inthe direction of the field from the large cross-section portion 1104 ofthe well into the small cross-section portion 1106 of the well, therebyconcentrating the sample in the area of the RF sense coil 1108. In thisexample, aggregates occupying a volume of approximately 1 μL areconcentrated into a volume of about 1 nL, thereby providing aconcentration of approximately 1000 times the original concentration.This results in an increase in sensitivity of the device by about 1000fold. The magnet(s) and/or magnetic field used to evoke an NMRrelaxation response is synergistically used to concentrate the targetanalyte for improved detection sensitivity. The device may include anarray of many micro wells and tiny RF coils surrounding the narrowportions 1106 of these wells.

FIG. 12 is a schematic diagram of a syringe analyte concentrator andassociated method 1200. This is an additional method for concentratingan analyte for improved sensitivity in the detection of the analyteusing the NMR device described herein. It may be used in combinationwith the magnetic concentrator shown in FIG. 11 and described above.However, the syringe analyte concentrator is not limited to applicationwith nanoparticle aggregation/NMR detection techniques.

In step 1210, a sample is drawn through a needle 1212 into a standard 1mL syringe 1214. In step 1220, the needle 1212 is removed and a testchamber 1222 is attached. The test chamber has a volume from about 10 toabout 400 μL and includes a molecular filter 1224 at the right side ofthe chamber. The molecular filter 1224 may be, for example, a membraneor molecular seive made from synthetic compounds, aluminosilicateminerals, clays, porous glass, microporous charcoal, activated carbon,desiccant, lime, silica gel, and/or zeolite. Various molecular filtersare available from suppliers such as the Pall Corporation, MilliporeCorporation, and Chromacol, for example. The molecular filter 1224 canbe used to concentrate DNA, viruses, proteins, and/or other analytes. Instep 1230, the test chamber 1222 is detached from the syringe 1214. Aplunger 1232 with an integral MEMS chip 1234 at the end having one ormore RF coil/well pairs is inserted. In step 1240, remaining fluid ispushed out through the filter 1224. In step 1250, the plunger 1232 ispulled back (to the left) about 1 mm to a detent, thereby drawing fluidheld up in the tip 1252 back through the filter 1224 and suspendingmolecules and nanoparticles. The test chamber 1222 can then be insertedinto the reader for NMR testing and analysis. Concentration of analytedepends upon chip size 1234 and syringe cross section. In general,because the MEMS chip is integral to the syringe plunger, the morewell/coil pairs in/on the chip, the greater the diameter and the lessconcentration obtained. For example, where there are 40 wells, with asyringe cross section of 40 mm² and 1 mm draw back (40 mm³ draw backvolume), the concentration is 25 times. Where there are 10 wells, with asyringe cross section of 10 mm² and 1 mm draw back (10 mm³ draw backvolume), the concentration is 100 times. Where there is one well, with asyringe cross section of 1 mm² and 1 mm draw back (1 mm³ draw backvolume), the concentration is 1000 times.

FIG. 13 is a schematic diagram of a membrane analyte concentrator 1300.This is an additional method for concentrating an analyte for improvedsensitivity in the detection of the analyte using the NMR devicedescribed herein. It may be used in combination with the magneticconcentrator shown in FIG. 11 and/or the syringe concentrator shown inFIG. 12 and described above.

The membrane analyte concentrator 1300 works by forcing ananalyte-containing liquid sample 1302 through a chamber 1304 containingnanoparticles 1306 (described herein) via a vacuum 1308. A molecularfilter 1310 keeps the molecules of interest in the chamber 1304, therebyconcentrating the analyte and improving performance. The chamber 1304shown in FIG. 13 has a length of about 500 μm. The molecular filter 1310may be a membrane with pores on the order of about 1 μm for detection ofa virus 1312 as analyte, for example, or with submicron pores for thedetection of DNA as the analyte.

NMR systems with RF coils and micro wells containing nanoparticlesensors described herein may be designed for detection and/orconcentration measurement of specific analyte(s) of interest bydevelopment of a model for particle aggregation phenomena and bydevelopment of an RF-NMR signal chain model. For example, experimentscan be conducted for analyte/nanoparticle systems of interest bycharacterizing the physics of particle aggregation, including, forexample, the effects of affinities, relevant dimensions, andconcentrations. Also, experiments can be conducted to characterize theNMR signal(s) (T2, T1, and/or other signal characteristics) as functionsof particle aggregation and magnetic particle characteristics. Signalcharacteristics specific to the MRS (magnetic resonance switch)phenomenon in a given system can be used to enhance detectionsensitivity and/or otherwise improve performance. The trade-off betweencertain design parameters affecting MRS-relaxation T2 (and/or T1)measurement performance may be determined via experimentation; forexample, trade offs between filling factor, coil geometries, Q factor,bandwidth, and/or magnetic bias field strength.

FIG. 14 is a schematic diagram of an electronics set-up 1400 for NMRmeasurement. The block diagram includes the basic circuit elements inthis configuration. The RF sensing coil(s) is/are represented at 1402.An RF pulse generator 1404 provides an RF pulse at or near Larmorfrequency. A single pulse may be delivered to the coil 1402, or a seriesof pulses may be delivered to the coil 1402 via switches. For example,enhanced sensitivity may be achieved for T2 relaxation measurementsusing multi-echo and/or spin-echo sequences. For example,Carr-Purcell-Meiboom-Gill (CPMG) fast spin-echo (FSE) sequences mayachieve greater T2 measurement sensitivity. The RF generator 1404 may ormay not be controlled by an off chip processor. A power splitter 1406and power combiner 1408 are shown in FIG. 14 for delivery of RFexcitation to the coil(s) 1402. The signal from each coil is amplifiedby an RF pre-amplifier 1410 and is processed by a mixer 1412, a low-passfilter 1414, and a low noise amplifier 1416 before signal analysis bythe off-chip processor 1408. The signal analysis processor 1408 mayalternatively be on-chip. The RF pre-amplifier is preferably in closeproximity to the respective coil(s) 1402. The signal analysis processor1408 manages the data and performs both time domain and frequency domainanalysis. Where there are multiple wells, a multiplexer could be used,for example, after conditioning by the RF pre-amplifier 1410. In certainembodiments, the RF coil(s) 1402, RF amplifier(s) 1410, and/or othercomponents shown in the diagram 1400 of FIG. 14 are micromachined, forexample, in a BiCMOS (or BiMOS) process, as a system-on-a-chip. BiCMOSrefers to the integration of bipolar junction transistors and CMOS(complementary-symmetry/metal-oxide semiconductor) technology into asingle device.

In order to maximize power transfer from the RF amplifier 1410, the coilis matched to a given impedance using variable capacitors. During signaldetection, the NMR signal from the coil 1402 may be amplified (e.g. by afactor of about 400) by the RF preamplifier 1410, and thendown-converted to audio-frequencies by the mixer 1412. The intermediatefrequency signal may be amplified (e.g. by a factor of about 100) andfiltered for frequencies, for example, above about 30 kHz before beingdigitized.

The NMR system may include a chip with RF coil(s) and electronicsmicromachined thereon. For example, the chip may be surfacemicromachined, such that structures are built on top of a substrate.Where the structures are built on top of the substrate and not insideit, the properties of the substrate are not as important as in bulkmicromachining, and expensive silicon wafers used in bulk micromachiningcan be replaced by less expensive materials such as glass or plastic.Alternative embodiments, however, may include chips that are bulkmicromachined. Surface micromachining generally starts with a wafer orother substrate and grows layers on top. These layers are selectivelyetched by photolithography and either a wet etch involving an acid or adry etch involving an ionized gas, or plasma. Dry etching can combinechemical etching with physical etching, or ion bombardment of thematerial. Surface micromachining may involve as many layers as isneeded.

Where the relaxation measurement is T2, accuracy and repeatability(precision) will be a function of the signal-to-noise ratio (S/N), thepulse sequence for refocusing (e.g. CPMG, BIRD, Tango, and the like), aswell as signal processing factors, such as signal conditioning (e.g.amplification, rectification, and/or digitization of the echo signals),time/frequency domain transformation, and signal processing algorithmsused. Signal-to-noise ratio may be a function of the magnetic bias field(B), sample volume, filling factor, coil geometry, coil Q-factor,electronics bandwidth, amplifier noise, and/or temperature, for example.

An illustrative experimental protocol for design or customization of ananalyte detection unit for detection of a particular analyte isdescribed below. The illustrative protocol includes performingexperiments with a single micro coil, for example, a solenoid wouldaround a capillary tube. Experiments would be conducted to determine howT2 changes as a function of analyte type and concentration, and NMRparticle ligand and affinity. Experiments would be conducted to analyzethe effect on the T2 signal of the excitation frequency (at and aroundthe Larmor frequency), the pulse sequence, signal conditioning, the biasfield (e.g. from about 0.45 T to about 7 T), and Q factor. The effect ofQ factor may be determined by performing experiments using coils madefrom different materials and/or performing and/or by performingexperiments at different temperatures, in order to test the effect ofcoil resistance on the signal quality.

For example, to obtain a 10-fold improvement in the 0.02 ng/mL detectionlimit for Troponin (10-fold increase in sensitivity), it would benecessary to discern a delta-T2 less than about 5.6 milliseconds from atraditional (non-MRS-measured) T2 of about 100 milliseconds. The minimumsignal-to-noise ratio (S/N) would need to be about 20 to detect thisdifference.

Assuming a target sample volume of 100 nl and a solenoid 542 micron indiameter by 400 micron long, the predicted performance is shown below inTable 1 in the shaded entry. This arrangement provides a predictedrobust S/N of 73 and a 0.3 microvolt signal with a 1 T field and 1000cps bandwidth. S/N increases to a predicted 1300 and 14 microvoltssignal with a 7 T field. Varying the coil design to create a higher Qmay enhance the performance further. Experiments can be performed athigher magnetic field strengths, e.g. a 7 T field strength provided bycommercially available NMR devices, to confirm the viability of systemdesign for achieving the 10-fold increase in the 0.02 ng/mL Troponindetection limit, or 56 femto-molar limit, with a 1 T field.

TABLE 1 Predicted coil performance Volume Volume Volume in c Coil DepthCoil Dia Filling Factor Bandwi Signal Coil Type in cc nanoliters micronmicrons micron Vs/Vc Turns Q cps Tesla S/N volts Solenoid 1 1,000,0001.00E+12 11730 10,000 1 1 10 1000 1 23,128 1.01E−03 0.1 100,000 1.00E+11280 20,468 1 1 10 1000 1 7,314 4.25E−03 0.01 10,000 1.00E+10 1000 3,4251 1 10 1000 1 2,313 1.19E−04 0.001 1,000 1.00E+09 5000 484 1 1 10 1000 22,069 9.51E−06 0.0001 100 1.00E+08 400 542 1 1 1 1000 1 73 2.97E−070.00001 10 1.00E+07 35 579 1 1 10 1000 1 73 3.40E−06 0.3 300,0003.00E+11 20000 4,195 0.044 3 10 1000 0.47 856 1.18E−04 0.04 40,0004.00E+10 20000 1,532 0.006 3 10 1000 0.47 114 1.58E−05 0.000002 22.00E+06 10 484 1 5 8 3E+05 1 2 9.51E−06 0.000001 1 1.00E+06 10 342 1 515 10 1 283 8.92E−06

EQUIVALENTS

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of detecting a cell surface protein in acell sample comprising: (i) obtaining a cell sample from a subject; and(ii) detecting the presence of the cell surface protein in the cellsample using a device, the device comprising: (a) a support defining awell for holding the cell sample and magnetic particles, the magneticparticles having binding moieties linked thereto, wherein the bindingmoieties are operative to alter an aggregation phenomena of the magneticparticles in the cell sample as a function of the presence orconcentration of cell surface protein in the cell sample; and (b) an RFcoil disposed about the well, the RF coil configured to detect an echoresponse produced by exposing the cell sample to a bias magnetic fieldcreated using one or more magnets and an RF excitation; wherein an echoresponse detected from the cell sample exposed to the bias magneticfield and RF excitation is indicative of detection of the one or moreanalytes in the cell sample, thereby detecting the presence of the cellsurface protein.
 2. The method of claim 1, wherein the cell surfaceprotein is a biomarker for cancer.
 3. The method of claim 1, wherein thebinding moieties are operative to bind an aggregation-inducing moleculein the cell sample, thereby producing an aggregate of multiply -linkedmagnetic particles and wherein the aggregate of multiply-linked magneticparticles is disaggregated as a function of the presence orconcentration of the cell surface protein in the cell sample.
 4. Adevice for the detection of a cell surface protein in a cell sample, thedevice comprising a support defining a well holding a cell samplecomprising magnetic particles and having an RF coil disposed about thewell, the RF coil configured to detect an echo response produced byexposing the liquid sample to a bias magnetic field created using one ormore magnets and an RF excitation, wherein the magnetic particles havebinding moieties on their surfaces, the binding moieties operative toalter an aggregation of the magnetic particles in the presence of thecell surface protein.
 5. The device of claim 4, wherein the well and theRF coil are configured to provide a filling factor of at least about0.1.
 6. The device of claim 4, wherein the well has a volume of lessthan about 300 μL.
 7. The device of claim 4, wherein the well and the RFcoil are configured such that the volume circumscribed by the RF coil isless than about 300 μL.
 8. The device of claim 4, wherein the RF coilhas a characteristic dimension from about 10 μm to about 1000 μm.
 9. Thedevice of claim 4, further comprising a tube for holding the cellsample, the tube having a varying cross section.
 10. The device of claim4, wherein the binding moieties are operative to bind anaggregation-inducing molecule in the cell sample, thereby producing anaggregate of multiply -linked magnetic particles and wherein theaggregate of multiply-linked magnetic particles is disaggregated as afunction of the presence or concentration of the cell surface protein inthe cell sample.